JP2011520108A - Assays for pathogenic conformers - Google Patents

Abstract

The present invention provides conditions under which a pathogenic conformer-specific binding reagent is bound to a sample suspected of containing a non-prion pathogenic conformer and, if present, to the pathogenic conformer. And, if there is a pathogenic conformer in the sample, detecting the presence of the pathogenic conformer by its binding to a reagent; and a non-prion pathogenic conformation in the sample A method is provided for detecting the presence of isomers, wherein pathogenic conformer-specific binding reagents are typically derived from prion protein fragments and interact preferentially with pathogenic prion proteins. A method of diagnosing conformational disease is also provided.

Description

(Cross-reference to related applications)
This application claims the benefit of US Provisional Application No. 61 / 049,396, filed Apr. 30, 2008, the contents of which are hereby incorporated by reference in their entirety. .

(background)
Protein conformation diseases include a wide variety of clinically unrelated diseases resulting from abnormal conformational changes of normal proteins to pathogenic conformers, such as infectious spongiform encephalopathy, Alzheimer's disease, ALS, and diabetes. Including. This change is then postulated to cause pathological conformer self-association and consequent tissue deposition, resulting in damage to surrounding tissue. These diseases share similarities in clinical symptoms, typically rapid progression from diagnosis to death after various lengths of incubation.

  Detection of pathogenic conformers of conformational disease proteins in living subjects and samples taken from living subjects has proven difficult. Current techniques for confirming the presence of pathogenic conformers in surviving patients are incomplete and invasive. For example, histopathological examination requires a dangerous biopsy for the subject. Histopathology is inherently susceptible to sampling errors as a lesion, and amyloid deposits can disappear depending on the extent to which the biopsy was taken. Therefore, definitive diagnosis and symptomatic treatment for these conditions prior to the subject's death remain a substantially unaddressed task.

  Deposition of amyloid beta protein (Aβ) aggregates, mainly Aβ1-40 (Aβ40) and 1-42 (Aβ42), is completely related to Alzheimer's disease (AD) and is a criterion for disease Is considered. However, the only definitive test for AD is immunohistochemical staining of Aβ plaques from postmortem brain samples. There are currently no FDA-approved pre-mortem diagnostic tests for AD. Plasma or CSF samples can be used for pre-mortem testing. Several pre-mortem AD tests focus on cerebrospinal fluid (CSF) and attempt to quantify soluble Aβ42. However, this biomarker serves only as an indirect measurement of AD.

  Tests that can specifically detect aggregated Aβ directly from other body fluids such as CSF or plasma have great advantages. Early detection of aggregated Aβ allows for a faster and more efficient diagnosis of Alzheimer's disease and evaluation of potential therapies.

  Tests that can detect pathogenic conformers of other conformational disease proteins are also desirable because they allow for a faster and more efficient diagnosis and evaluation of potential therapies for these conformational diseases.

(Concise overview of preferred embodiment)
The present invention relates in part to pathogenic conformer-specific binding reagents that interact preferentially with both pathogenic prion proteins and other non-prion pathogenic conformers. In some embodiments, the PCSB reagent is PrP19-30 (SEQ ID NO: 242), PrP23-30 (SEQ ID NO: 243), PrP100-111 (SEQ ID NO: 244), PrP101-110 (SEQ ID NO: 245), PrP154-165 ( Derived from prion protein fragments such as SEQ ID NO: 246) and PrP226-237 (SEQ ID NO: 247). In other embodiments, the pathogenic conformer specific binding reagent has the amino acid sequence of: SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, and SEQ ID NO: 247.

  In other embodiments, the PCSB reagent is a peptoid analog of a prion protein fragment. In some embodiments, the peptoid analog has the following structure:

の１つを有する。

One of them.

  In preferred embodiments, the pathogenic conformer specific binding reagent has a net positive charge of at least 3 at physiological pH or at least 4 at physiological pH.

  In one aspect, a method for detecting the presence of a non-prion pathogenic conformer is provided. Detection methods include binding of a pathogenic conformer-specific binding reagent to a sample suspected of containing a non-prion pathogenic conformer and, if present, to the non-prion pathogenic conformer. Contacting to form a complex; and if there is a non-prion pathogenic conformer in the sample, by its binding to a pathogenic conformer specific binding reagent, Detecting a non-prion pathogenic conformer, wherein the pathogenic conformer-specific binding reagent is preferentially interacting with the pathogenic prion protein from the prion protein fragment.

  The non-prion pathogenic conformer detected by the reagent can be a conformer associated with an amyloid disease such as systemic amyloidosis, tauopathy, or synucleinopathy. For example, the non-prion pathogenic conformer may be associated with Alzheimer's disease, ALS, immunoglobulin-related disease, serum amyloid A-related disease, or type II diabetes.

  In a preferred embodiment, the non-prion pathogenic conformer detected with the PCSB reagent is an Alzheimer's disease conformer, such as amyloid-β or tau protein. In such cases, preferred pathogenic conformer specific binding reagents are prion fragments PrP19-30 (SEQ ID NO: 242), PrP23-30 (SEQ ID NO: 243), PrP100-111 (SEQ ID NO: 244), PrP101-110. (SEQ ID NO: 245), PrP154-165 (SEQ ID NO: 246), PrP226-237 (SEQ ID NO: 247), SEQ ID NO: 14, SEQ ID NO: 50, or SEQ ID NO: 68,

を含む。

including.

  Samples to be tested are organs, whole blood, blood fractions, blood components, plasma, platelets, serum, cerebrospinal fluid (CSF), brain tissue, nervous system tissue, muscle tissue, bone marrow, urine, tears, non-neural system It can be a tissue, biopsy or autopsy. In preferred embodiments, the sample is plasma or cerebrospinal fluid.

  In the methods of the invention, the pathogenic conformer specific binding reagent is typically detectably labeled, eg, with biotin. The reagent is typically bound to a solid support such as nitrocellulose, polystyrene latex, polyvinyl fluoride, diazotized paper, nylon membrane, activated beads, and magnetically responsive beads.

  In another embodiment, other methods for detecting the presence of non-prion pathogenic conformers are also provided. The method involves binding a sample suspected of containing a non-prion pathogenic conformer with a pathogenic conformer-specific binding reagent and, if present, the non-prion pathogenic conformer. Contacting under conditions that allow for formation of a complex; contacting the complex with a conformation disease protein-specific binding reagent under conditions that permit binding; and non-prion in the sample Detecting the presence of a non-prion pathogenic conformer by its binding to a conformational disease protein-specific binding reagent, if present, and Isomer-specific binding reagents are derived from prion protein fragments and interact preferentially with pathogenic prion proteins. The conformational disease protein-specific binding reagent can be a labeled antibody. When the non-prion pathogenic conformer is an Aβ protein, the conformation disease protein-specific binding reagent is an anti-Aβ antibody.

  In one embodiment, the method further comprises removing unbound sample material after complex formation.

  Other methods for detecting the presence of a non-prion pathogenic conformer include a sample suspected of containing a non-prion pathogenic conformer, if present with a pathogenic conformer-specific binding reagent. Contacting under conditions that permit binding of the reagent to the non-prion pathogenic conformer to form a first complex; removing unbound sample material; and non-prion pathogenicity Dissociating the conformer from the first complex, thereby providing a dissociated non-prion pathogenic conformer; the dissociated non-prion pathogenic conformer is a conformational disease protein Contacting with a specific binding reagent under conditions that allow binding to form a second complex; and if there is a non-prion pathogenic conformer in the sample, By detecting formation Detecting the presence of a non-prion pathogenic conformer, wherein the pathogenic conformer-specific binding reagent is preferentially interacting with the pathogenic prion protein from the prion protein fragment. To do.

  The formation of the second complex can be detected using a detectably labeled second conformation disease protein-specific binding reagent.

  In some embodiments, the pathogenic conformer specific binding reagent and / or conformational disease protein specific binding reagent is coupled to a solid support.

  Non-prion pathogenic conformers can be obtained from the first complex (with PCSB reagent) by exposing the complex to guanidine thiocyanate, exposing the complex to sodium hydroxide, or increasing the complex. Exposure to pH or low pH can in some cases cause dissociation by neutralizing the high or low pH after dissociation.

  In embodiments where the non-prion pathogenic conformer is Aβ, the protein is preferably complexed by exposure to high pH conditions such as sodium hydroxide, preferably at about 0.1 N NaOH at about 80 ° C. Is dissociated from.

  Another method for detecting the presence of a non-prion pathogenic conformer is to use a sample suspected of containing a non-prion pathogenic conformer as a first pathogenic conformer specific binding reagent. Contacting, if present, under conditions that allow binding of the first reagent to a non-prion pathogenic conformer to form a first complex; A sample suspected of containing an isomer is transferred to a second pathogenic conformer-specific binding reagent containing a detectable label and a non-prion pathogenic conformer in the first complex. Contacting under conditions that allow binding of the second reagent; and if there is a non-prion pathogenic conformer in the sample, its binding to the second reagent results in non-prion pathogenic conformer Detecting a body; and comprising at least a first disease and a second disease Sex conformer specific binding reagent is derived from prion protein fragments, preferentially interact with pathogenic prion protein.

  Another method for detecting the presence of a non-prion pathogenic conformer includes: (a) treating a sample suspected of containing a non-prion pathogenic conformer with a conformation disease protein-specific binding reagent; Contacting, if present, under conditions that allow binding of the CDPSB reagent to a non-prion pathogenic conformer to form a complex; (b) removing unbound sample material; (C) the complex can be combined with a pathogenic conformer specific binding reagent containing a detectable label and a pathogenic conformer specific binding reagent to a non-prion pathogenic conformer. Contacting with a pathogenic conformer-specific binding reagent, if there is a non-prion pathogenic conformer in the sample, thereby binding the non-prion pathogenic conformer Detecting step; Has even without pathogenic conformer specific binding reagent is derived from prion protein fragments, preferentially interact with pathogenic prion protein.

  Yet another method for detecting the presence of a non-prion pathogenic conformer includes providing a solid support comprising a pathogenic conformer specific binding reagent; and the solid support is detectably labeled. The binding affinity of the pathogenic conformer-specific binding reagent to the detectably labeled ligand is weaker than the binding affinity to the non-prion pathogenic conformer, And, when a non-prion pathogenic conformer is present in the sample, binding the sample to a solid support under conditions that allow it to bind to the reagent and displace the ligand; Detecting a complex formed between the prion pathogenic conformer and the pathogenic conformer specific binding reagent derived from the prion protein fragment, Preferentially interact with sex prion protein.

  Also provided are methods for distinguishing between non-prion pathogenic conformers and non-prion non-pathogenic conformers. The method involves binding a pathogenic conformer-specific binding reagent to a sample suspected of containing a non-prion pathogenic conformer and, if present, binding of the reagent to a non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex; and binding to the pathogenic conformer to the reagent to form a non-prion pathogenic conformer and a non-prion non-pathogenic conformer And the pathogenic conformer specific binding reagent is preferentially interacting with the pathogenic prion protein from the prion protein fragment.

  A method of diagnosing non-prion conformation disease is also provided. The method involves binding a pathogenic conformer-specific binding reagent to a sample suspected of containing a non-prion pathogenic conformer and, if present, binding of the reagent to a non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex; and if there is a non-prion pathogenic conformer in the sample, detect the non-prion pathogenic conformer by its binding to a reagent And diagnosing conformation disease when a non-prion pathogenic conformer is detected, wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment. Interact preferentially with pathogenic prion proteins.

  In another embodiment, the present invention provides a pathogenic conformer-specific binding reagent in a sample suspected of containing a non-prion pathogenic conformer, and a non-prion pathogenic conformer, if present. Contacting under conditions that allow binding of the reagent to the complex; and if there is a non-prion pathogenic conformer in the sample, to the pathogenic conformer specific binding reagent And detecting a non-prion pathogenic conformer by its binding; a pathogenic conformer specific binding reagent is SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, Methods are provided for detecting the presence of non-prion pathogenic conformers, including peptoid regions comprising 236, 237, 238, 239, 240, or 241.

  In yet another aspect, the invention provides a pathogenic conformer-specific binding reagent in a sample suspected of containing a non-prion pathogenic conformer, and a non-prion pathogenic conformation, if present. Contacting under conditions that allow binding of the reagent to the isomer to form a complex; and if there is a non-prion pathogenic conformer in the sample, pathogenic conformer specific binding Detecting a non-prion pathogenic conformer by its binding to the reagent; a pathogenic conformer specific binding reagent:

から選択される、非プリオン病原性配座異性体の存在を検出する方法を提供する。

To detect the presence of a non-prion pathogenic conformer.

FIGS. 1A and 1B show the accumulation of misfolded Aβ40 and Aβ42 in a panel of Alzheimer samples but not normal samples, and that these misfolded Aβ peptides are only recognized after denaturation by the Aβ ELISA. Treat 10% (w / v) brain homogenate (BH) from normal or Alzheimer's disease individuals with either water (natural, white bars) or 5.4M GdnSCN (denatured, black bars) for 30 minutes at room temperature. And then diluted with TBST (50 mM Tris, pH 7.5; 150 mM NaCl; 0.05% Tween-20) so that 100 nL of 10% BH per 100 μL of sample was added to each ELISA well. ELISA capture plates were coated at 2.5 μg / mL with (1A) 11A50-B10 antibody (specific for the C-terminus of Aβ40) or (1B) 12F4 antibody (specific for the C-terminus of Aβ42). After incubation at 37 ° C. for 1 hour, the plate was washed 4 times with TBST and the bound Aβ peptide was diluted with TBST + 0.1% bovine serum albumin alkaline phosphatase (AP) conjugated 4G8 detection antibody (residual Aβ The group 17-24 was recognized) at 0.2 μg / ml. After 1 hour at 37 ° C., the plate was again washed 4 times with TBST. LumiphosPlus was a chemiluminescent substrate. Patient identification numbers for normal (320, 326, 327, 328) and Alzheimer patients (remaining numbered samples) are as indicated. A buffer control (bkgd) was used to determine the ELISA background signal. FIG. 2 shows that misfolded Aβ42 is an insoluble aggregated form that can be pelleted by centrifugation, and that the solubility is caused by denaturation of the aggregate and detection by the described ELISA is performed. 100 nl of 10% BH was treated with either water (natural, white bar) or 5.4 M GdnSCN (denatured, black bar) for 30 minutes at room temperature before dilution with 100 μl TBST. One set of samples was directly used for sandwich ELISA (all samples). Another set was centrifuged at 135,520 g for 1.5 hours at 4 ° C. The pellet fraction was denatured in 6M GdnSCN for 30 minutes at room temperature and diluted with 100 μl TBST. Both supernatant and pellet fractions were then utilized for ELISA. Aβ42 peptide was captured with 12F4 antibody and detected with 4G8-AP detection antibody as described above. FIG. 3 shows that PSR1 binding (ie pull-down) of Aβ42 aggregates in plasma can be attributed to peptoid XIIb rather than beads. 75 nL of 10% BH from normal or Alzheimer's disease patients was spiked into 100 μL of 80% plasma in TBSTT (50 mM Tris, pH 7.5; 150 mM NaCl; 1% Tween-20; 1% Triton-X100). The BH spike solution was incubated with either PSR1 or negative beads (GLUT) for 1 hour at 37 ° C. and then washed 5 times with TBST. The capture material was denatured with 6M GdnSCN for 30 minutes at room temperature, diluted with TBST, and then applied to a 12F4 (recognizing C-terminus of Aβ42) capture plate. Capture material was detected by 4G8-AP as described above. Patient identification numbers for normal (320, 326, 327, 328) and Alzheimer patients (remaining numbered samples) are as indicated. A buffer control (bkgd) was used to determine the ELISA background signal. FIGS. 4A and 4B show endogenous soluble Aβ40 and Aβ42 levels in normal human plasma detected by ELISA. Concentrated pool human plasma was diluted with TBST buffer and applied to 11A50-B10 (4A) or 12F4 (4B) plates to capture Aβ40 and Aβ42, respectively. Captured peptides were detected with 4G8-AP detection antibody as described above. FIG. 5 (AF) shows that PSR1 binds Aβ40 and Aβ42 aggregates but does not recognize Aβ aggregates solubilized by denaturing agents. FIG. 5A is a standard curve of Aβ42 generated by applying denatured synthetic Aβ42 to a 12F4 coated ELISA plate. FIG. 5B is the detection of Aβ42 from a dose-escalating natural or modified Alzheimer 10% BH (patient number 291). BH is treated with water (natural, white triangles) or 5.4M GdnSCN (denatured, gray circles) for 30 minutes at room temperature, then diluted with 100 μl TBST and loaded onto a 12F4 (specific to C-terminus of Aβ42) capture plate Applied to assess the level of Aβ in BH. FIG. 5C shows 30% treatment with either water (natural, white circles and triangles) or 5.4M GdnSCN (denatured, gray circles and triangles) for 30 minutes before 80% human plasma in TBSTT buffer. Shows the amount of Aβ42 captured from Alzheimer 10% BH (patient number 291) diluted at 100 μl and incubated with either PSR1 (triangle) or negative (GLUT, circle) beads for 1 hour at 37 ° C. . After pull-down, the beads were washed 5 times with TBST and the bound material was eluted with 6M GdnSCN for 30 minutes at room temperature. Samples were diluted with TBST and applied to 12F4 capture plates. Capture material was detected by 4G8-AP as described above. FIG. 5D is a standard curve of Aβ40 generated by denaturing various concentrations of synthetic Aβ40 and applying it to an 11A50-B10 coated (specific to the C-terminus of Aβ40) ELISA capture plate. FIG. 5E shows Alzheimer's 10% BH (patient number 291) treated with water (natural, white triangle) or 5.4M GdnSCN (denatured, gray circle) for 30 minutes at room temperature and then diluted with 100 μl TBST. The amount of Aβ40 detected is shown. Samples were applied directly to the 11A50-B10 capture plate to assess the level of Aβ42 in BH. FIG. 5F shows treatment with either water (natural, white circles and triangles) or 5.4M GdnSCN (denatured, gray circles and triangles) for 30 minutes at room temperature, followed by 80% human plasma in TBSTT buffer. Shown is the amount of Aβ40 captured from Alzheimer's 10% BH (patient number 291) diluted in 100 μl and incubated for 1 hour at 37 ° C. with either PSR1 or negative (GLUT) beads. After pull-down, the beads were washed 5 times with TBST and the bound material was eluted with 6M GdnSCN for 30 minutes at room temperature. Samples were diluted in TBST and applied to 11A50-B10 capture plates. Capture material was detected by 4G8-AP as described above. FIG. 5 (AF) shows that PSR1 binds Aβ40 and Aβ42 aggregates but does not recognize Aβ aggregates solubilized by denaturing agents. FIG. 5A is a standard curve of Aβ42 generated by applying denatured synthetic Aβ42 to a 12F4 coated ELISA plate. FIG. 5B is the detection of Aβ42 from a dose-escalating natural or modified Alzheimer 10% BH (patient number 291). BH is treated with water (natural, white triangles) or 5.4M GdnSCN (denatured, gray circles) for 30 minutes at room temperature, then diluted with 100 μl TBST and loaded onto a 12F4 (specific to C-terminus of Aβ42) capture plate Applied to assess the level of Aβ in BH. FIG. 5C shows 30% treatment with either water (natural, white circles and triangles) or 5.4M GdnSCN (denatured, gray circles and triangles) for 30 minutes before 80% human plasma in TBSTT buffer. Shows the amount of Aβ42 captured from Alzheimer 10% BH (patient number 291) diluted at 100 μl and incubated with either PSR1 (triangle) or negative (GLUT, circle) beads for 1 hour at 37 ° C. . After pull-down, the beads were washed 5 times with TBST and the bound material was eluted with 6M GdnSCN for 30 minutes at room temperature. Samples were diluted with TBST and applied to 12F4 capture plates. Capture material was detected by 4G8-AP as described above. FIG. 5D is a standard curve of Aβ40 generated by denaturing various concentrations of synthetic Aβ40 and applying it to an 11A50-B10 coated (specific to the C-terminus of Aβ40) ELISA capture plate. FIG. 5E shows Alzheimer's 10% BH (patient number 291) treated with water (natural, white triangle) or 5.4M GdnSCN (denatured, gray circle) for 30 minutes at room temperature and then diluted with 100 μl TBST. The amount of Aβ40 detected is shown. Samples were applied directly to the 11A50-B10 capture plate to assess the level of Aβ42 in BH. FIG. 5F shows treatment with either water (natural, white circles and triangles) or 5.4M GdnSCN (denatured, gray circles and triangles) for 30 minutes at room temperature, followed by 80% human plasma in TBSTT buffer. Shown is the amount of Aβ40 captured from Alzheimer's 10% BH (patient number 291) diluted in 100 μl and incubated for 1 hour at 37 ° C. with either PSR1 or negative (GLUT) beads. After pull-down, the beads were washed 5 times with TBST and the bound material was eluted with 6M GdnSCN for 30 minutes at room temperature. Samples were diluted in TBST and applied to 11A50-B10 capture plates. Capture material was detected by 4G8-AP as described above. FIG. 5 (AF) shows that PSR1 binds Aβ40 and Aβ42 aggregates but does not recognize Aβ aggregates solubilized by denaturing agents. FIG. 5A is a standard curve of Aβ42 generated by applying denatured synthetic Aβ42 to a 12F4 coated ELISA plate. FIG. 5B is the detection of Aβ42 from a dose-escalating natural or modified Alzheimer 10% BH (patient number 291). BH is treated with water (natural, white triangles) or 5.4M GdnSCN (denatured, gray circles) for 30 minutes at room temperature, then diluted with 100 μl TBST and loaded onto a 12F4 (specific to C-terminus of Aβ42) capture plate Applied to assess the level of Aβ in BH. FIG. 5C shows 30% treatment with either water (natural, white circles and triangles) or 5.4M GdnSCN (denatured, gray circles and triangles) for 30 minutes before 80% human plasma in TBSTT buffer. Shows the amount of Aβ42 captured from Alzheimer 10% BH (patient number 291) diluted at 100 μl and incubated with either PSR1 (triangle) or negative (GLUT, circle) beads for 1 hour at 37 ° C. . After pull-down, the beads were washed 5 times with TBST and the bound material was eluted with 6M GdnSCN for 30 minutes at room temperature. Samples were diluted with TBST and applied to 12F4 capture plates. Capture material was detected by 4G8-AP as described above. FIG. 5D is a standard curve of Aβ40 generated by denaturing various concentrations of synthetic Aβ40 and applying it to an 11A50-B10 coated (specific to the C-terminus of Aβ40) ELISA capture plate. FIG. 5E shows Alzheimer's 10% BH (patient number 291) treated with water (natural, white triangle) or 5.4M GdnSCN (denatured, gray circle) for 30 minutes at room temperature and then diluted with 100 μl TBST. The amount of Aβ40 detected is shown. Samples were applied directly to the 11A50-B10 capture plate to assess the level of Aβ42 in BH. FIG. 5F shows treatment with either water (natural, white circles and triangles) or 5.4M GdnSCN (denatured, gray circles and triangles) for 30 minutes at room temperature, followed by 80% human plasma in TBSTT buffer. Shown is the amount of Aβ40 captured from Alzheimer's 10% BH (patient number 291) diluted in 100 μl and incubated for 1 hour at 37 ° C. with either PSR1 or negative (GLUT) beads. After pull-down, the beads were washed 5 times with TBST and the bound material was eluted with 6M GdnSCN for 30 minutes at room temperature. Samples were diluted in TBST and applied to 11A50-B10 capture plates. Capture material was detected by 4G8-AP as described above. FIGS. 6A and 6B show the capture profiles of various pathogenic conformer specific binding reagents (6 different peptides and PSR1) for PrP ^{Sc in} vCJD samples, AD samples containing buffer or 50% plasma. compared to capture profiles of Aβ aggregates in, thereby indicating that the PSR1 and prion-derived peptides bind to PrP ^Sc and Aβ aggregates of buffer and plasma. Biotinylated peptide was coated onto M280-streptavidin beads and then incubated with the sample. PSR1 peptoid was coupled to Dynal M270-carboxylic acid beads. For vCJD experiments, 100 nL of 10% vCJD BH was diluted with 100 μL of TBSTT (8A, upper panel) or 50% plasma in TBSTT (8B, upper panel) for 1 hour with the indicated pathogenic conformer specific binding reagent. And incubated at 37 ° C. The beads were washed 6 times with TBST and eluted with 0.1 M NaOH for 10 minutes at room temperature. The eluent is neutralized with 0.3M NaH ₂ PO ₄ for 5 minutes at room temperature and then applied to a 3F4 coated capture plate (2.5 μg / mL 3F4 antibody, recognizing residues 109-112 of the human PrP sequence) did. The material was captured for 1 hour at 37 ° C., washed 6 times with TBST, and detected with AP-conjugated POM2 antibody (recognizing prion octarepeat sequence) in 0.01 × casein blocker in TBST. After 1 hour incubation at 37 ° C, the plates were washed again and detected with LumiphosPlus substrate. In AD experiments, 50 nL of 10% AD BH was similarly spiked into TBSTT (8A, lower panel) or 50% plasma during TBSTT (8B, lower panel). Samples were similarly pulled down with a conformer specific binding reagent, eluted with 6M GdnSCN for 30 minutes at room temperature and diluted with TBST. The eluent was applied to a 12F4 capture plate and detected by horseradish peroxidase (HRP) conjugated 4G8. The vCJD data is from Lau, A. et al. L, et al., Proc Natl Acad Sci USA. 104 (28): 11551-11556 (2007). FIG. 7 shows the same panel of prion-derived peptides and PSR1 capture profiles for aggregated Aβ42 from AD BH spiked to 50% CSF in TBSTT. Pull-down and Aβ42 specific sandwich ELISA detection was performed as described above. FIG. 8 shows NaOH concentration titration and temperature screening for optimization of Aβ42 denaturation in AD brain homogenate versus normal brain homogenate (NBH). FIG. 9, Panels AF illustrate exemplary peptoid substitutions that can be made to prepare any of the PCSB reagents described herein. Peptoids are circled in each panel, and the exemplary reagents described herein (QWNKPSKPKTNG, sequence, in which the proline residue (residue 8) is replaced by an N-substituted glycine (peptoid) residue. No. 250). Panel A shows a peptide reagent in which the proline residue is replaced by a peptoid residue: N- (S)-(1-phenylethyl) glycine; Panel B, a peptide in which the proline residue is replaced by a peptoid residue Reagents are shown: N- (4-hydroxyphenyl) glycine; Panel C shows peptide reagents in which the proline residue is replaced by a peptoid residue: N- (cyclopropylmethyl) glycine; Panel D shows the proline residue Shows peptide reagent substituted with peptoid residue: N- (isopropyl) glycine; Panel E shows peptide reagent with proline residue substituted with peptoid residue: N- (3,5-dimethoxybenzyl) Glycine; and Panel F shows peptide reagents in which the proline residue is replaced by a peptoid residue. : N- butyl glycine. FIG. 10 shows the structure of an exemplary PEG-conjugated pathogenic conformer-specific binding reagent described herein. FIG. 11 shows that Aβ capture by PSR1 and PrP23-30 is superior to capture by β sheet blockers. AL30 is an Aβ20-16 reverse sequence (using D-amino acids instead of L-amino acids) and has the following structure: biotin-AHX-D (FFVLK) -CONH2 (SEQ ID NO: 252). AL32 is Aβ20-16 and has the following structure: biotin-AHX-FFVLK-CONH2 (SEQ ID NO: 253). AL33 is Aβ16-20-NmeL and has the following structure: biotin-AHX-KLVFF-NmeL-CONH2 (SEQ ID NO: 254). NmeL is a “standard” amino acid modified N-methylated lysine available from most peptide custom synthesis companies. AL34 is Aβ (16-20-NmeL) ₂ and has the following structure: biotin-AHX-KLVFF-NmeL-AHX-KLVFF-NmeL-CONH2 (SEQ ID NO: 255). FIG. 12 shows that significant levels of total tau are detected in both normal and Alzheimer brains. Normal brain (patient ID numbers 320, 326, 327, 328) or AD brain (patient ID numbers 325, 334, 325, 264-1, 230-1, 218-2, 221-1, 201-2, 184-1) 177-1, and 291) Homogenates were either untreated (natural, white bars) or treated with 3M GnSCN (black bars). Total tau was quantified using the BioSource Tawi immunoassay kit. FIG. 13 shows the amount of tau that specifically binds to PSR1 beads as opposed to control glutathione beads. Normal brain (patient ID numbers 320, 326, 327, 328) or AD brain (patient ID numbers 325, 334, 325, 264-1, 230-1, 218-2, 221-1, 201-2, 184-1) 177-1, and 291) Homogenates were diluted with buffer and incubated with either M270-glutathione (white bar) or PSR1 (black bar). FIG. 14 shows that PSR1 binds tau aggregates but does not recognize tau aggregates solubilized by denaturing agents. Normal brain (patient ID number 320, 326) or AD brain (patient ID number 334, 230) is treated with either water (N) or 5M GdnSCN (D) and diluted with 25% plasma in TBSTT, It was then incubated with either M270 glutathione control beads (white bars) or PSR1 (black bars). After pulling down, the beads were washed with TBSTT and incubated with GdnSCN. Captured tau was quantified using a BioSource Tawi immunoassay kit. FIGS. 15A and B show the data used to calculate the LOD for the monomeric soluble Aβ sandwich ELISA and the aggregated Aβ PSR1 pull-down. In FIG. 15A, varying amounts of synthetic soluble Aβ (pg / mL) are detected by sandwich ELISA. In FIG. 15B, various amounts of 10% AD brain homogenate spiked into 200 ul of pooled normal human CSF are subjected to PSR1 pulldown and detected by sandwich ELISA. A black circle represents Aβ40. White circles represent Aβ42. FIGS. 15A and B show the data used to calculate the LOD for the monomeric soluble Aβ sandwich ELISA and the aggregated Aβ PSR1 pull-down. In FIG. 15A, varying amounts of synthetic soluble Aβ (pg / mL) are detected by sandwich ELISA. In FIG. 15B, various amounts of 10% AD brain homogenate spiked into 200 ul of pooled normal human CSF are subjected to PSR1 pulldown and detected by sandwich ELISA. A black circle represents Aβ40. White circles represent Aβ42. FIG. 16 compares the total amount of Aβ42 aggregates in AD BH (squares) with Aβ42 aggregates bound to PSR1 (triangles). FIG. 17 shows the effect of increasing plasma concentration on the binding of monomeric Aβ42 (triangles) and aggregated Aβ42 (circles). FIG. 18 compares the signals of NBH (normal brain homogenate, white bar) and AD (brain homogenate from Alzheimer's disease patient, black bar) at various dissociation conditions. FIG. 19 shows the data used to calculate the LOD for ELISA and PSR1 bead pulldown for all tau (A & B), P-tau 231 (C & D) and P-tau 181 (E & F). FIG. 19A shows a tau ELISA standard curve. FIG. 19B shows tau pulldown with AD BH spike CSF (200 ul assay). FIG. 19C shows a P-tau 231 ELISA standard curve. FIG. 19D shows P-tau 231 pull-down with AD BH spike CSF (70 uL CSF). FIG. 19E shows a P-tau 181 ELISA standard curve. FIG. 19F shows P-tau 181 pulldown with AD BH spike CSF (70 uL CSF).

(Short description of the table)
Table 1 shows exemplary conformational diseases and related conformational disease proteins.

  Table 2 shows additional conformational disease proteins and related conformational diseases.

  Table 3 shows exemplary peptide sequences used to generate PCSB reagents.

  Table 4 shows exemplary peptoid regions suitable for generating PCSB reagents.

  Table 5 shows the abbreviated keys used in Table 4.

  Table 6 gives the related structure of each sequence shown in Table 4.

  Table 7 quantifies the pull-down efficiency of PSR1.

  Table 8 quantifies the tau levels measured in Example 10.

  Table 9 quantifies the tau levels measured in Example 11.

  Table 10 quantifies the binding of Aβ40 and 42 to PSR1 beads from CSF of individuals who are not Alzheimer's disease.

  Table 11 quantifies PSR1 monomericity and binding to aggregated Aβ in the presence of increasing plasma concentrations.

(Short description of sequence list)
SEQ ID NOs: 1-11 provide the amino acid sequences of prion proteins from different species: human (SEQ ID NO: 1), mouse (SEQ ID NO: 2), human (SEQ ID NO: 3), Syrian hamster (hamster) (SEQ ID NO: 4 ), Cattle (SEQ ID NO: 5), sheep (SEQ ID NO: 6), mouse (SEQ ID NO: 7), elk (SEQ ID NO: 8), fallow deer (fallow) (SEQ ID NO: 9), mule deer (mava) (SEQ ID NO: 10), and white-tailed deer (white) (SEQ ID NO: 11).

  SEQ ID NOs: 12-228 provide the amino acid sequences of exemplary peptide sequences used to generate PCSB reagents.

  SEQ ID NOs: 229-241 provide modified amino acid sequences of exemplary peptoid regions used to generate PCSB reagents.

  SEQ ID NOs: 242-249 provide the amino acid sequences of exemplary prion protein fragments used to generate PCSB reagents.

  SEQ ID NO: 250 provides the amino acid sequence of an exemplary peptide sequence used to generate a PCSB reagent.

  SEQ ID NO: 251 provides amino acid sequence residues 19-30 of the human prion protein as shown in SEQ ID NO: 1.

  SEQ ID NOs: 252-255 provide the amino acid sequences of the beta sheet breakers AL30, AL32, AL33, and AL34.

  SEQ ID NOs: 256-261 provide the amino acid sequences of the modified prion protein fragments tested in Example 3.

(Detailed description of the invention)
The present invention preferentially interacts with pathogenic conformers of other conformational diseases such as Alzheimer's disease, diabetes, systemic amyloidosis, etc. It relates to the surprising and unexpected discovery of interacting. These PCSB reagents are typically derived from prion protein fragments.

  The discovery that these PCSB reagents also interact preferentially with non-prion pathogenic conformers makes detection using these PCSB reagents for non-prion conformational diseases and conformational disease proteins and prion-related diseases It enables the development of assays, diagnostic assays, and purification or isolation methods.

Without wishing to be bound by any theory, the ability of these PCSB reagents to preferentially bind non-prion pathogenic conformers and detect them is common to certain pathogenic conformers. This may be due to the presence of a structural motif. Lau, A., et al., Incorporated herein by reference as if included herein. L et al., Proc Natl Acad Sci USA. 104 (28): 11551-11556 (2007) suggests that the interaction between the PCSB reagent derived from the prion protein fragment and PrP ^Sc is highly dependent on the positive charge. The interaction does not appear to be affected by scrambling the sequence, but the properties of individual amino acids other than the positive charge also seem to play some role in the interaction. These studies suggest that these PCSB reagents bind structural motifs rather than the linear sequence domain of PrP ^Sc associated with disease.

Many pathogenic conformers that form amyloid share similar physical properties. ^{PrP Sc} for example, pathogenic conformer of the prion protein, the following characteristics: beta increased sheet content (from about 3% in PrP ^C in ^{PrP Sc>} 40%) indicates, ^{PrP Sc} fibers , Composed of β-sheets oriented vertically along the fiber axis. Applicants have shown that binding to one of the structural motifs common to all amyloidogenic proteins is preferentially associated with a pathogenic conformer of a non-prion protein that interacts preferentially with the pathogenic prion protein. I think it is a mechanism of interaction.

  These PCSB reagents need not be part of a larger structure or other type of backbone molecule to show such preferential interactions with pathogenic conformers. While not wishing to be bound by any particular theory, these PCSB reagents have conformations that allow binding to pathogenic conformers but not binding to non-pathogenic conformers. It seems to take voluntarily. The exemplified PCSB reagents provide a starting point (eg, with respect to size or sequence characteristics) for PCSB reagents useful in the methods of the invention, but more desirable attributes (eg, higher affinity, higher stability, higher It will be apparent to those skilled in the art that many modifications can be made to produce PCSB reagents with solubility, lower protease sensitivity, higher specificity, easier synthesis, and the like.

  In general, the PCSB reagents described herein can interact preferentially with pathogenic conformers. Therefore, with these reagents, for example, immediate detection of the presence of pathogenic conformers, and therefore survival, by directing disease-aggregating proteins to a state that is organized, aggregated or otherwise detectable. Diagnosis of pathogenic conformers in essentially any biological or non-biological sample, including blood and spleen as well as living or dead brain, spinal cord, cerebrospinal fluid or other neural tissue systems It becomes possible. PCSB reagents are useful for a wide range of isolation, purification, detection, diagnostic and therapeutic applications.

  PCSB reagents used in the methods of the invention are described in further detail in WO05 / 016137 and WO07 / 030804, which are incorporated herein by reference.

  The practice of the present invention uses conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology within the skill of the art, unless otherwise indicated. Such techniques are explained fully in the literature. For example Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (.. S.Colowick and N.Kaplan, eds, Academic Press, Inc); and Handbook of Experimental Immunology, Vols . I-IV (DM Weir and CC Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook et al. , KS Ed., CRC Press, 1997); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. Eds., 1999, John Wiley &Sons); Molecular Biology Technologies: An Intensive Laboratory Course, (Ream et al., 1998, Academic Press) u (Int. (Newton & Graham eds., 1997, Springer Verlag); Peters and Dalrymple, Fields Virology (2d ed), Fields et al. (Eds.), B. et al. N. See Raven Press, New York, NY.

  It is to be understood that the reagents and methods of the present invention are not limited to a particular formulation or process parameter and can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not limiting.

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

(I. Definition)
To facilitate an understanding of the present invention, selected terms used in this application are discussed below.

  As used herein, the term “pathogenic” can mean that a protein or conformer actually causes disease, or the term “pathogenic” refers to a protein or conformer. It may simply mean that the body is associated with the disease and is therefore present when the disease is present. Thus, a pathogenic protein or conformer as used in connection with the present disclosure is not necessarily a protein that is a specific causative agent of the disease and thus may or may not be infectious. The term “pathogenic conformer” is used more specifically to refer to a conformation of a protein associated with a disease and / or a β-sheet rich conformation. A “pathogenic conformer” refers to a protein associated with a disease, regardless of whether its conformation is a misfolded conformer, an aggregated misfolded conformer, or a mixture of the two. Arbitrary conformation. The terms “non-pathogenic” and “cellular”, when used in reference to a conformational disease protein or conformer, refer to the normal conformer of a protein whose presence is not associated with a disease. Used interchangeably. Pathogenic conformers associated with certain diseases, such as Alzheimer's disease, may be referred to as “pathogenic Alzheimer's disease conformers”.

  The term “pathogenic conformer-specific binding reagent” or “PCSB reagent” is not limited to this, but is contrasted with non-pathogenic conformers due to increased affinity or specificity. Refers to any type of reagent, including peptides and peptoids, that preferentially interact with pathogenic conformers. Preferential interactions do not necessarily require an interaction between specific amino acid residues and / or motifs of each peptide. For example, in certain embodiments, a pathogenic conformer specific binding reagent described herein interacts preferentially with a pathogenic conformer but nevertheless a non-pathogenic conformer. It may also be possible to bind isomers at a weak but detectable level (eg, 10% or less of the binding exhibited by the polypeptide of interest). Typically, weak binding, ie background binding, can be readily distinguished from preferential interactions with the pathogenic conformer of interest, for example by use of appropriate controls. In general, pathogenic conformer specific binding reagents used in the methods of the invention bind pathogenic conformers in the presence of excess non-pathogenic forms.

  A pathogenic conformer-specific binding reagent "interacts" when it binds with another peptide or protein specifically, non-specifically, or in some combination of specific and non-specific binding. It is said. A reagent is “preferred” when it binds a pathogenic conformer with a higher affinity and / or higher specificity for a pathogenic conformer than for a non-pathogenic conformer. To interact with. " "Interact preferentially", "preferentially interact", "bind selective", "selectively bind", And the term “selectively capture” is used interchangeably herein. It should be understood that preferential interactions do not necessarily require an interaction between specific amino acid residues and / or motifs of each peptide. For example, in certain embodiments, the PCSB reagents described herein interact preferentially with pathogenic conformers but nevertheless weakly detect non-pathogenic conformers. It may be possible to bind at a certain level (eg, 10% or less of the binding exhibited by the polypeptide of interest). Typically, weak binding, ie background binding, can be easily distinguished from preferential interactions with the compound or polypeptide of interest, eg by use of appropriate controls. In general, the reagents described herein bind pathogenic conformers in the presence of a non-pathogenic conformer in excess of 100-fold.

  PCSB reagents utilized in the methods described herein are derived from prion protein fragments and interact preferentially with pathogenic forms of prion protein. The term “derived from a prion protein fragment” as used herein refers to a reagent having a chemical structure based on the chemical structure of the prion protein fragment. Such a reagent can be a peptide fragment having a natural prion protein sequence, a peptide fragment having a natural prion protein sequence with conservative amino acid substitutions, or a peptoid analog of a peptide fragment of a prion protein.

  The term “derived from a prion protein fragment and preferentially interacts with a pathogenic prion protein”, as used herein, refers to a reagent having a combination of the properties defined above. As an example, such a reagent has a chemical structure based on the chemical structure of a prion protein fragment as defined above, and has a higher affinity for pathogenic prion proteins than for non-pathogenic prion proteins. And / or bind with high specificity.

  The term “non-prion pathogenic conformer” as used herein is not related to prion diseases as defined herein, but is a pathogenic conformer of a conformational disease protein. Point to.

“Conformational disease protein” refers to a conformational disease in which the structure of the protein has been altered (eg, misfolded) to produce an abnormal conformation such as undesirable fibrillar or amyloid polymerization in the context of β-pleated sheets. Refers to pathogenic and non-pathogenic conformers of related proteins. Examples of conformational disease proteins include, without limitation, Alzheimer's disease proteins such as Aβ and tau; prion proteins such as PrP ^Sc and PrP ^C , and the diabetic protein amylin. Below a non-limiting list of diseases associated with proteins that assume two or more different conformations,

に示す。
「コンフォメーション病タンパク質」は本明細書で使用するように、本明細書に記載する配列と同じ配列を有するポリペプチドに限定されない。この用語が、同定済みのまたは未同定の種または疾患（たとえばアルツハイマー病、パーキンソン病など）のいずれによるコンフォメーション病タンパク質も含むことは、容易に明らかとなる。当業者は、本開示および当該分野の教示を考慮して、たとえば配列比較プログラム（たとえばＢＬＡＳＴおよび本明細書に記載する他のもの）または構造特性もしくはモチーフの同定およびアライメントを使用して、任意の他のプリオンタンパク質の図に示した配列に対応する領域を決定することができる。

Shown in
A “conformational disease protein”, as used herein, is not limited to a polypeptide having the same sequence as that described herein. It will be readily apparent that this term includes conformational disease proteins from any identified or unidentified species or disease (eg, Alzheimer's disease, Parkinson's disease, etc.). Those skilled in the art will be able to use any sequence comparison program (eg, BLAST and others described herein) or identification and alignment of structural features or motifs in light of the present disclosure and the teachings of the art. Regions corresponding to the sequences shown in other prion protein diagrams can be determined.

  A “conformational disease protein-specific binding reagent” or “CDPSB reagent” is any molecule that preferentially interacts with a particular conformational disease protein, as opposed to other conformational disease proteins and other types of proteins. Refers to a type of reagent. Preferably, the conformational disease protein specific binding reagent binds to both pathogenic and non-pathogenic conformers of the conformational disease protein. However, in many instances, conformational disease protein-specific binding reagents can only bind to a soluble form of the conformational disease protein, and thus cannot bind aggregate / misfold pathogenic conformers. In this case, it may be necessary to denature the insoluble pathogenic conformer to be detected. Typically such reagents are monoclonal or polyclonal antibodies.

The terms “prion”, “prion protein”, “PrP protein” and “PrP” are different from pathogenic conformers (scrapie protein, pathogenic protein form, pathogenic isoform, pathogenic prion and PrP ^Sc. referred to) and non-pathogenic conformer (cellular protein form, cellular isoform, nonpathogenic isoform, nonpathogenic prion protein, and PrP ^C and are different called) of both, of course, pathogenic con It is used interchangeably herein to refer to denatured forms and various recombinant forms of prion protein that may not have either conformation or normal cell conformation. Pathogenic conformers are associated with human and animal disease states (spongiform encephalopathy). Non-pathogenic conformers are usually present in animal cells and can be converted to pathogenic PrP ^Sc conformation under appropriate conditions. Prions are naturally produced in a wide variety of mammalian species including humans, sheep, cows and mice. A representative amino acid sequence of human prion protein is shown as SEQ ID NO: 1. A representative amino acid sequence of mouse prion protein is shown as SEQ ID NO: 2. Other representative sequences are provided as SEQ ID NOs: 3-11. A fragment of a prion protein is designated by a SEQ ID NO corresponding to the actual sequence or by indicating the amino acid position of the first and last amino acids of the fragment. Unless indicated otherwise, the fragment referred to by indicating the first and last amino acids of the fragment is based on the sequence of the human prion protein as shown by SEQ ID NO: 1. For example, the term “PrP _19-30 ” refers to a peptide having the sequence of LGLCKKKRPKPGG (SEQ ID NO: 251).

  The term “Alzheimer's disease (AD) protein” or “AD protein” refers to pathogenic conformers (pathogenic protein forms, pathogenic isoforms, pathogenic Alzheimer's disease proteins, and Alzheimer's disease conformers in various ways. Both) and non-pathogenic conformers (normally referred to as normal cell morphology, non-pathogenic isoforms, variously referred to as non-pathogenic Alzheimer's disease proteins), either pathogenic or normal cell conformations Are also used interchangeably herein to refer to denatured forms and various recombinant forms of Alzheimer's disease protein that may not have. Exemplary Alzheimer's disease proteins include Aβ and tau proteins.

  The terms “amyloid beta”, “amyloid-β”, “A beta”, “Aβ”, “Aβ42”, “Aβ40”, and “Aβ40 / 42” are all used herein as amyloid precursors. It refers to amyloid-β peptide, which is a 39-43 amino acid long fragment formed by cleavage of body protein (APP). The term Aβ is used to refer generally to any form of amyloid-β peptide. The term “Aβ42” refers to the fragment corresponding to amino acids 1-42 of APP. The term “Aβ40” refers to the fragment corresponding to amino acids 1-40 of APP. The term Aβ40 / 42 is used to refer to both Aβ40 and Aβ42 isoforms.

  The term “diabetic protein” refers to pathogenic conformers (pathogenic protein forms, pathogenic isoforms, variously called pathogenic diabetes proteins) and non-pathogenic conformers (normal cell forms, non-pathogenic) To refer to denatured forms of diabetic proteins and various recombinant forms that may not have either pathogenic or normal cell conformations, as well as isoforms, variously called non-pathogenic diabetic proteins) As used herein. An exemplary type II diabetes protein is amylin, also known as islet amyloid polypeptide (IAPP).

  “Fragment” as used herein refers to an intact full-length protein and a peptide consisting only of a portion of a structure as found in nature. For example, the fragment can include a C-terminal deletion and / or an N-terminal deletion of the protein. Typically, a fragment retains one, some or all of the functions of the full-length polypeptide sequence from which it is derived. Typically, the fragment comprises at least 5 contiguous amino acid residues of the natural protein; preferably at least about 8 contiguous amino acid residues; and more preferably at least about 10, 11, 12, 13, 14, 15, Includes 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acid residues.

  “Isolated” when referring to a polynucleotide or polypeptide is that the indicated molecule is found in nature, separated from the whole organism and distinct, or polynucleotide or polypeptide Is not found in nature, it means that the polynucleotide or polypeptide is sufficiently free of other biological macromolecules that it can be used for its intended purpose.

  “Peptoid” is generally used to refer to a peptidomimetic containing at least one, preferably two or more amino acid substitutions, preferably N-substituted glycines. Peptoids are described in particular in US Pat. No. 5,811,387. As used herein, a “peptoid reagent” is a molecule having an amino terminal region, a carboxy terminal region, and at least one “peptoid region” between the amino terminal region and the carboxy terminal region. The amino terminal region refers to the region on the amino terminal side of the reagent that typically does not contain any N-substituted glycines. The amino terminal region can be H, alkyl, substituted alkyl, acyl, amino protecting group, amino acid, peptide, and the like. The carboxy-terminal region refers to the carboxy-terminal region of the peptoid that does not contain any N-substituted glycines. The carboxy terminal region can include H, alkyl, alkoxy, amino, alkylamino, dialkylamino, carboxy protecting groups, amino acids, peptides, and the like.

  A “peptoid region” is a region that includes the N-substituted glycine starting from the N-substituted glycine closest to the amino terminus and ending with the N-substituted glycine closest to the carboxy terminus. The peptoid region generally refers to the portion of the reagent in which at least three of the amino acids therein are replaced by N-substituted glycines.

  “Physiologically relevant pH” refers to a pH of about 5.5 to about 8.5; or about 6.0 to about 8.0; or usually about 6.5 to about 7.5.

  “Aliphatic” refers to a straight-chain or branched hydrocarbon moiety. Aliphatic groups can include heteroatoms and carbonyl moieties.

  “Alkyl”, whether used alone or as part of another group, refers to an aliphatic hydrocarbon chain, including but not limited to, unless expressly specified otherwise. , 1-6, 1-5, 1-4, or 1-3 straight and branched chain containing carbon atoms. For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl and the like are included in the term “alkyl”.

  “Alkenyl” includes, but is not limited to, for example, vinyl, allyl, 2-methyl-allyl, 4-but-3-enyl, 4-hex-5-enyl, 3-methyl-but-2- intended to indicate an alkyl group containing at least one double bond, such as enyl, etc., for example 2-7, 2-6, 2-5, or 2-4 carbon atoms To do.

  “Alkynyl” refers to an alkyl group having at least one triple carbon-carbon bond, such as 2-7, 2-6, 2-5, or 2-4 carbon atoms. Intended. Exemplary alkynyl groups include ethynyl, propynyl and the like.

  “Alkoxy”, whether used alone or as part of another group, has the ordinary meaning of a group of formula —O-alkyl, such as methoxy, where alkyl is as defined herein. As defined in.

  “Halo” or “halogen”, when used alone or as part of another group, has the ordinary meaning of a Group VII element, such as F, Cl, Br, and I.

  “Aryl”, when used alone or as part of another group, is, for example, 1, 2 or 3 rings, such as 6-20, 6-14, or 6-10 ring carbons. Aromatic aromatic hydrocarbon systems such as phenyl, benzyl, naphthyl, naphthalene, anthracene, phenanthrenyl, anthracenyl, pyrenyl and the like are meant. The definition of aryl also includes aromatic systems containing one or more fused non-aromatic carbocyclyl or heterocyclyl rings, such as 1,2,3,4-tetrahydronaphthalene and indane. An aryl group containing a fused non-aromatic ring can be attached via an aromatic or non-aromatic moiety.

  “Aryl-alkyl” or “aralkyl” means a group of the formula -alkyl-aryl, where aryl and alkyl have the definitions herein.

  “Aryloxy” has the usual meaning of a group of formula —O-aryl, eg, hydroxyphenyl, where aryl is as defined herein.

  “Aralkoxy” has the usual meaning of a group of formula —O-alkyl-aryl, for example methoxyphenyl, wherein alkoxy and aryl are as defined herein.

  “Cycloalkyl”, whether used alone or as part of another group, is a cyclic alkyl, alkenyl, or alkynyl group, for example a monocyclic, bicyclic, of 3-10 carbon atoms, Tricyclic, fused, bridged or spiro-saturated hydrocarbon moieties such as cyclopropyl have their usual meanings. The term “cycloalkyl-aryl” is intended to indicate a group of formula -aryl-cycloalkyl, where aryl and cycloalkyl are as defined herein. “Cycloalkylalkyl” is intended to indicate a group of formula -alkyl-cycloalkyl, such as cyclopropylmethyl or cyclohexylmethyl, where alkyl and cycloalkyl are as defined herein. is there.

  As used herein, a “heteroaryl” group refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (eg, having 2, 3 or 4 fused rings) systems. Examples of heteroaryl groups include, without limitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, Including pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl and the like. In some embodiments, the heteroaryl group has 1 to about 20 carbon atoms, and in further embodiments about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.

  As used herein, “heterocycloalkyl” includes cyclized alkyl, alkenyl, alkynyl groups in which one or more of the ring-forming carbon atoms has been replaced by a heteroatom such as an O, N, or S atom. , Refers to a non-aromatic heterocycle. Exemplary “heterocycloalkyl” groups are morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl Pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl and the like. The definition of heterocycloalkyl includes moieties having one or more aromatic rings fused to a non-aromatic heterocyclic ring (ie, having a common bond) such as phthalimidyl, naphthalimidyl, and heterocycles such as indolene and isoindolene groups. Also included are ring benzo derivatives. In some embodiments, the heterocycloalkyl group has 1 to about 20 carbon atoms, and in further embodiments about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double or triple bonds.

  “Heteroarylalkyl” refers to a radical of the formula -alkyl-heteroaryl, where alkyl and heteroaryl are as defined herein.

  “Acyl” refers to a radical of the formula —C (O) -alkyl. In some embodiments, the acyl group has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms.

  “Aminoacyl” refers to a radical of the formula —C (O) -alkyl-amino where alkyl is as defined herein.

  “Alkylamino” refers to a radical of the formula —NH-alkyl, where alkyl is as defined herein.

“Dialkylamino” refers to a radical of the formula —N (alkyl) ₂ where alkyl is as defined herein.

  “Haloalkyl” refers to an alkyl group substituted by one or more halogens, where alkyl and halogen are as defined herein.

  “Alkoxyalkyl” refers to a radical of the formula -alkyl-alkoxy, where alkyl and alkoxy are as defined herein.

  “Carboxyalkyl” refers to a radical of the formula -alkyl-COOH where alkyl is as defined herein.

“Carbamyl” refers to a radical of the formula —C (O) NH ₂ .

“Carbamylalkyl” refers to a radical of the formula -alkyl-C (O) NH ₂ , where alkyl is as defined herein.

“Guanidinoalkyl” refers to a radical of the formula -alkyl-NHC (═NH) NH ₂ , where alkyl is as defined herein.

  “Thiol” refers to a group of formula —SH.

  “Alkylthiol” refers to a radical of the formula —S-alkyl, where alkyl is as defined herein.

  “Alkylthioalkyl” refers to a radical of the formula -alkly-S-alkyl, where alkyl is as defined herein.

  “Imidazolylalkyl” refers to a radical of the formula -alkyl-imidazolyl, where alkyl is as defined herein.

  “Piperidylalkyl” refers to a radical of the formula -alkyl-piperidinyl, where alkyl is as defined herein.

  “Naphthylalkyl” means a radical of the formula -alkyl-naphthyl, for example (8′-naphthyl) methyl, where naphthyl has its usual meaning and alkyl is as defined herein.

  “Indolylalkyl” means a radical of the formula -alkyl-indole, such as 3′-indolylethyl, and 3′-indolylmethyl, where indole has its usual meaning and alkyl is as defined herein. As defined in the book.

  “N-containing heterocyclyl” is intended to refer to any heteroaryl or heterocycloalkyl group containing at least one ring-forming N atom. Exemplary N-containing heterocyclyl groups include pyridinyl, imidazolyl, piperidinyl, piperazinyl, pyrrolyl, indolyl, and the like.

  “N-containing heterocyclylalkyl” is intended to refer to an alkyl substituted by an N-containing heterocyclylalkyl.

“Amino” and “primary amino” refer to NH ₂ . “Secondary amino” refers to NHR and “tertiary amino” refers to NR ₂ where R is any suitable substituent.

“Ammonium” is intended to refer to the group —N (R) ³⁺ , where R can be any suitable moiety such as alkyl, cycloalkyl, aryl, cycloalkylalkyl, arylalkyl, and the like.

  “Amino acid” refers to any of the 20 naturally occurring and genetically encoded α-amino acids or protected derivatives thereof. A protected derivative of an amino acid can contain one or more protecting groups in the amino moiety, the carboxy moiety, or the side chain moiety. Examples of amino protecting groups are formyl, trityl, phthalimide, trichloroacetyl, chloroacetyl, bromoacetyl, iodoacetyl, and urethane type blocking groups such as benzyloxycarbonyl, 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl , 3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, t-butoxycarbonyl, 2- (4-xe L) -Isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl, 2- (p-toluyl) ) -Prop-2-yloxycarbonyl, cyclopentanyloxy-carbonyl, 1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 2- (4-Toluylsulfonyl) -ethoxycarbonyl, 2- (methylsulfonyl) ethoxycarbonyl, 2- (triphenylphosphino) -ethoxycarbonyl, fluorenylmethoxycarbonyl (“FMOC”), 2- (trimethylsilyl) ethoxy Carbonyl, allyloxycarbonyl, 1- (trimethylsilylmethyl) prop-1-enyloxycarbonyl, 5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-ethynyl- 2-propoxycarbonyl, cyclopropylmethoxycarbonyl, 4- (decycloxy) benzyloxycarbonyl, isobornyloxycarbonyl, 1-piperidyloxycarbonyl, etc .; benzoylmethylsulfonyl group, 2-nitrophenylsulfenyl, diphenylphosphine oxide and the like Contains an amino protecting group. Examples of carboxy protecting groups are methyl, p-nitrobenzyl, p-methylbenzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2, 4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4'-dimethoxybenzhydryl, 2,2 ', 4,4'-tetramethoxybenzhydryl, t- Butyl, t-amyl, trityl, 4-methoxytrityl, 4,4′-dimethoxytrityl, 4,4 ′, 4 ″ -trimethoxytrityl, 2-phenylprop-2-yl, trimethylsilyl, t-butyldimethylsilyl, Phenacyl, 2,2,2-trichloroethyl, beta- (di (n-butyl) methylsilyl) ethyl, p- Including ruenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl, 1- (trimethylsilylmethyl) prop-1-en-3-yl and similar moieties.The type of protecting group used depends on the rest of the molecule. It is not important as long as the derivatized protecting group can be selectively removed at the appropriate place without destruction, further examples of protecting groups are described in E. Haslam, Protecting Groups in Organic Chemistry, (JGW McOmie). , Ed., 1973), at Chapter 2; and TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, (1991), at Chapter 7, Its entirety is incorporated herein by reference.

“N-substituted glycine” refers to a residue of formula — (NR—CH ₂ —CO) —, wherein each R is (C ₂ -C ₆ ) alkyl, halo (C ₁ -C ₆ ) alkyl, ( C ₂ -C _{6) alkenyl, (C} 2 -C _{6) alkynyl,} (C 6 _{-C 10)} cycloalkyl - aryl, amino _(C 1 -C ₆₎ alkyl, ammonium _(C 1 -C ₆₎ alkyl, hydroxy (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy (C ₁ -C ₆ ) alkyl, carboxy, carboxy (C ₂ -C ₆ ) alkyl, carbamyl, carbamyl (C ₂ -C ₆ ) alkyl, guanidino, guanidino _(C 1 -C ₆₎ alkyl, amidino, amidino _(C 1 -C ₆₎ alkyl, _{thiol, (C} 1 -C ₆₎ alkylthiol, 2-10 alkylthio carbon atoms Alkyl, N-containing heterocyclyl, N-containing heterocyclyl _(C 1 -C ₆₎ alkyl, imidazolyl, of 4 to 10 carbon atoms imidazolylalkyl, piperidyl, carbon atoms 5-10 piperidyl alkyl, indolyl, carbon atoms 9 to 15 Non-hydrogen moieties such as indolylalkyl, naphthyl, naphthylalkyl of 11 to 16 carbon atoms, and aryl (C ₁ -C ₆ ) alkyl; wherein each R moiety is a halogen Optionally substituted with 1 to 3 substituents independently selected from hydroxy, and (C ₁ -C ₆ ) alkoxy.

_{- (NR-CH 2 -CO)} - In some embodiments of, R _{represents, (C} 2 -C ₆₎ alkyl, halo _(C 1 -C _{6) alkyl, (C} 2 -C ₆₎ alkenyl, ( C ₂ -C _{6) alkynyl,} (C 6 _{-C 10)} cycloalkyl - aryl, amino _(C 1 -C ₆₎ alkyl, hydroxy _(C 1 -C _{6) alkyl, (C} 1 -C ₆₎ alkoxy (C ₁ -C ₆₎ alkyl, carboxy, carboxy _(C 2 -C ₆₎ alkyl, carbamyl, carbamyl _(C 2 -C ₆₎ alkyl, guanidino, guanidino _(C 1 -C ₆₎ alkyl, _{thiol, (C} 1 -C ₆₎ alkylthiol, of 2 to 10 carbon atoms alkylthioalkyl, imidazolyl, of 4 to 10 carbon atoms imidazolylalkyl, piperidyl, 5-10 carbon atoms Peri Jill alkyl, indolyl, carbon atoms 9 to 15 amino indolylalkyl, naphthyl, carbon atoms 11 to 16 amino naphthylalkyl, diphenyl _(C 1 -C ₆₎ alkyl or aryl _(C 1 -C ₆₎ alkyl Wherein each R moiety is optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxy, and (C ₁ -C ₆ ) alkoxy.

_{- (NR-CH 2 -CO)} - In some embodiments of, R represents substituted halogen, by hydroxy or _(C 1 -C _{6) 1~3} substituents independently selected from alkoxy (C ₂ -C ₆ ) alkyl, amino (C ₁ -C ₆ ) alkyl, hydroxy (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy (C ₁ -C ₆ ) alkyl, guanidino ( C ₁ -C ₆ ) alkyl, indolylalkyl having 9 to 15 carbon atoms, naphthylalkyl having 11 to 16 carbon atoms, diphenyl (C ₁ -C ₆ ) alkyl or aryl (C ₁ -C ₆ ) alkyl. .

_{- (NR-CH 2 -CO)} - In some embodiments of, R is charged moiety at physiologically relevant pH. Examples of R positively charged at physiologically relevant pH include, for example, amino (C ₁ -C ₆ ) alkyl, ammonium (C ₁ -C ₆ ) alkyl, guanidino, guanidino (C ₁ -C ₆ ) alkyl, Including amidino, amidino (C ₁ -C ₆ ) alkyl, N-containing heterocyclyl, and N-containing heterocyclyl (C ₁ -C ₆ ) alkyl, wherein each R moiety is halogen, C1-C3 methoxy, and C1-C3 alkyl Optionally substituted by 1 to 3 substituents independently selected from

_{- (NR-CH 2 -CO)} - In some embodiments of, R is part of the neutral at physiologically relevant pH. Examples of neutral R at physiologically relevant pH are, for example, (C ₂ -C ₆ ) alkyl, halo (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C _{6) alkynyl,} (C 6 _{-C 10)} cycloalkyl - _{aryl, (C} 1 -C ₆₎ alkoxy _(C 1 -C ₆₎ alkyl, 2 to 10 carbon atoms alkylthioalkyl, diphenyl _(C 1 -C ₆ ) Alkyl, and aryl (C ₁ -C ₆ ) alkyl. Further examples are ethyl, prop-1-yl, prop-2-yl, 1-methylprop-1-yl, 2-methylprop-1-yl, 3-phenylprop-1-yl, 3-methylbutyl, benzyl, 4- Includes chloro-benzyl, 4-methoxy-benzyl, 4-methyl-benzyl, 2-methylthioeth-1-yl, and 2,2-diphenylethyl.

In some embodiments of — (NR—CH ₂ —CO) —, R is amino (C ₁ -C ₆ ) alkyl (eg, aminobutyl).

  Further exemplary N-substituted glycines are those in which R is ethyl, prop-1-yl, prop-2-yl, 1-methylprop-1-yl, 2-methylprop-1-yl, 3-phenylprop-1-yl, 3 -Methylbutyl, benzyl, 4-hydroxybenzyl, 4-chloro-benzyl, 4-methoxy-benzyl, 4-methyl-benzyl, 2-hydroxyethyl, mercaptoethyl, 2-aminoethyl, 3-propionic acid, 3-aminopropyl 4-aminobutyl, 2-methylthioeth-1-yl, carboxymethyl, 2-carboxyethyl, carbamylmethyl, 2-carbamylethyl, 3-guanidinoprop-1-yl, imidazolylmethyl, 2,2-diphenyl Including those that are ethyl or indol-3-yl-ethyl.

  Also included are salts, esters, and protected forms of N-substituted glycines (eg, those that are N-protected, such as by Fmoc or Boc).

  Methods for generating amino acid substituents including N-substituted glycines are disclosed in US Pat. No. 5,811,387, specifically incorporated herein by reference in its entirety.

  “Monomer” or “subunit” refers to a molecule, such as a peptide, that can be linked to another monomer to form a chain. Amino acids and N-substituted glycines are exemplary monomers. When combined with other monomers, the monomers can be referred to as “residues”.

(II. Conformational disease)
The present invention relates to methods for detecting pathogenic conformers of non-prion conformation disease proteins and methods for diagnosing diseases associated with such proteins. The conformational disease proteins and their corresponding diseases are listed in the table below.

に示す疾患を含む。

Including the diseases shown in

  The conformational diseases of the present invention include any disease associated with proteins that form two or more different conformations other than those associated with prions. Diseases of particular interest herein include amyloid diseases that all exhibit cross-beta sheet characteristics, such as Alzheimer's disease, systemic amyloidosis, tauopathy, and synucleinopathies. Other diseases of interest are diabetes and polyglutamine disease.

  In certain embodiments of the methods of the invention, conformational disease protein-specific binding reagents (“CDPSB reagents”) are used to either capture or detect both non-pathogenic and pathogenic conformers. Is done. The particular CDPSB reagent used will depend on the pathogenic conformer being detected. For example, if the conformation disease to be diagnosed is Alzheimer's disease, then the CDPSB reagent can be an antibody that recognizes both non-pathogenic and pathogenic conformers of Alzheimer's disease protein Aβ.

(III. Reagents used in the method of the present invention)
The pathogenic conformer specific binding reagent ("PCSB reagent") used in the present invention is a reagent that interacts preferentially with pathogenic prion protein.

  Typically, the PCSB reagent is derived from a prion protein fragment. Preferably, such PCSB reagents are either modified peptides, including those commonly known as peptides or peptoids.

In certain embodiments, such PCSB reagents are polycationic. Most preferably, the PCSB reagent has a net positive charge of at least 3 or 4 at physiological pH. Without wishing to be bound by theory, Applicants believe that the PCSB reagents described herein are non-prion pathogenic conformations by a mechanism similar to the mechanism by which PCSB reagents derived from prion protein fragments bind to PrP ^Sc. It is believed that it binds to isomers and thus exhibits similar binding properties. Lau et al. (Cited herein above) show that the core peptide sequence required for binding to PrP ^Sc has four positively charged amino acids in both plasma and buffer. Peptides containing only three positively charged amino acid residues can bind PrP ^Sc only in buffer. Furthermore, alanine scanning of peptides that bind PrP ^Sc in both plasma and buffer is reduced to background levels by removing any one of the positively charged amino acids.

(A. Preferred prion protein fragment)
The PCSB reagent is preferably derived from the amino acid sequence of a prion protein fragment. These preferred regions are exemplified for both the mouse prion sequence (SEQ ID NO: 2) and the human prion sequence (SEQ ID NO: 1). The PCSB reagent used in the method of the invention is preferably derived from a prion protein fragment that acts as a basis for other PCSB reagents known to interact preferentially with pathogenic prion proteins, but the resulting PCSB reagent is As long as it can interact preferentially with pathogenic prion protein, it may be derived from any prion protein fragment. Particularly preferred sequences are described below.

  The PCSB reagent used in the present invention can be derived from a fragment of the amino acid sequence of any species or variant. The polynucleotide and amino acid sequences of prion proteins produced by many different species including human, mouse, sheep and cow are known. Variations of these sequences are also present in each species. For example, in certain embodiments, the peptide PCSB reagents described herein are derived from any of the sequences set forth in SEQ ID NOs: 1-11. Although the sequences of PCSB reagents specifically disclosed herein are based on either mouse or human prion sequences, those skilled in the art will readily substitute corresponding sequences from other species, where appropriate. be able to.

  The preferred length of the prion protein fragment from which the PCSB reagent can be derived is a residue of 3-5 in length, a residue of 6-10 (or any integer in between), a length of 11-20 Residues (or any integer in between), residues 21-75 (or any integer in between), residues 75-100 (or any integer in between), or lengths Including polypeptides with more than 100 residues. Preferably, the peptide is about 3-100 residues in length. In general, one of ordinary skill in the art can readily select the maximum length in view of the teachings herein. In addition, reagents such as those described herein, eg, synthetic peptides, can include additional molecules such as labels, linkers, or other chemical moieties (eg, amyloid specific dyes such as biotin, control red or thioflavin). Such moieties may further improve the interaction of the PCSB reagent with the pathogenic conformer and / or further detection of the pathogenic conformer.

In a preferred embodiment, the PCSB reagent is a prion protein fragment known to interact preferentially with pathogenic prion protein, such as the following human prion protein fragments: PrP _19-30 (SEQ ID NO: 242), PrP _23-30 (SEQ ID NO: _{243), PrP 100-111} (SEQ ID NO: _{244), PrP 101-110} (SEQ ID NO: _{245), PrP 154-165} (SEQ ID NO: _{246), PrP 226-237} (SEQ ID NO: 247), SEQ ID NO: 14, Derived from fragments having sequences corresponding to SEQ ID NO: 50 and SEQ ID NO: 68.

(B. PCSB peptide reagent)
A PCSB reagent derived from a prion protein fragment can have the exact amino acid sequence of the prion protein fragment or can be a modified or modified form of the prion protein fragment. The PCSB reagent used in the method of the present invention is preferably derived from a prion protein fragment that acts as a basis for other PCSB reagents known to interact preferentially with the pathogenic prion protein, but the resulting reagent is pathogenic. As long as it can interact preferentially with the sex prion protein, it may be derived from any prion protein fragment.

  PCSB reagents include derivatives of the amino acid sequence of prion protein fragments having one or more substitutions, additions and / or deletions, including one or more unnatural amino acids. Preferably, the derivative has at least about 50% identity, preferably at least about 70% identity to any wild type or reference sequence, more preferably any wild type described herein. Or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to the reference sequence Shows sequence identity. Sequence (or percent) identity can be determined using any method known to those of skill in the art, such as those described below. Such derivatives can include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like.

  Techniques for determining amino acid sequence similarity or percent identity are well known in the art. In general, “similarity” is an amino acid-to-amino acid comparison of two or more polypeptides at the appropriate location where the amino acids are identical or have similar chemical and / or physical properties such as charge or hydrophobicity. It means having. And so-called “percent identity” can be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity are also well known in the art, determining the nucleotide sequence of a gene's mRNA (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby. And comparing this to a second amino acid sequence. In general, “identity” refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.

  Two or more amino acid or polynucleotide sequences can be compared by determining their “percent identity”. “Percent identity” refers to the sequence between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence and multiplying the result by 100. It can be determined by direct comparison of information (reference sequence and sequence with unknown% identity to the reference sequence). For peptide analysis, Advances in Appl. Math. 2: 482-489, 1981, to the local homology algorithm of Smith and Waterman, ALIGN, Dayhoff, M .; O. in Atlas of Protein Sequence and Structure M. in Atlas of Protein Sequence. O. Dayhoff ed. , 5 Suppl. 3: 353-358, National biomedical Research Foundation, Washington, DC, and other readily available computer programs can be used for analysis. Programs that determine nucleotide sequence identity, such as the BESTFIT, FASTA, and GAP programs that also rely on the Smith and Waterman algorithm, are available from Wisconsin Sequence Analysis Package, Version 8 (Genetics Computer Group, I. Madison, Id.). These programs are easily utilized by the default parameters recommended by the producer and described in the Wisconsin Sequence Analysis Package mentioned above. For example, the percent identity of a particular nucleotide sequence with respect to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a 6 nucleotide site gap penalty.

Another method of establishing percent identity in the context of the present invention is the copyright of University of Edinburgh, John F. Collins and Shane S. It is to use the MPSRCH ^™ package program developed by Sturok and available from many sources, for example the Internet. From this set of packages, the default parameters are used in the scoring table (eg, gap open penalty 12, gap extension penalty 1, and gap 6), and the Smith-Waterman algorithm can be used. From the generated data, the “Match” value reflects “sequence identity”. Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences Sort = HIGH SCORE; database = non-duplicate, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

  PCSB reagents can also include PCSB reagents with modifications to the native prion protein sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the polypeptide retains the desired activity. In certain embodiments, conservative amino acid substitutions are preferred. A conservative amino acid substitution is a substitution associated with its side chain that occurs within a family of amino acids. Genetically encoded amino acids are generally classified into four families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, Leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarity = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. For example, an isolated substitution of threonine and serine with a glutamine salt of aspartic acid with leucine and isoleucine or valine, or a similar conservative substitution with an amino acid structurally related to an amino acid can On the other hand, it can be reasonably predicted that there will be no significant effect. Such modifications can be deliberate, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or errors due to PCR amplification. In addition, the following effects: reduced toxicity; increased affinity and / or specificity for pathogenic conformers; enhanced cell processing (eg, secretion, antigen presentation, etc.); and to B cells and / or T cells Modifications can be made that have one or more of facilitating presentation.

  PCSB reagents include one or more analogs of amino acids (eg, including non-natural amino acids, etc.), both natural and non-natural (eg, synthetic), as well as peptides having substituted linkages in the art. Other modifications known in can also be included. Synthetic peptides, dimers, multimers (eg, tandem repeats, multiantigenic peptide (MAP) forms, linear binding peptides), cyclizations, branched molecules, and the like are therefore considered peptides. This also includes molecules that contain one or more N-substituted glycine residues (“peptoids”) and other synthetic amino acids or peptides. (For a description of peptoids, see, eg, US Pat. Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al. (2000) Chem Biol. 7 (7): 463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89 (20): 9367-9371).

For a general review of these and other amino acid analogs and peptidomimetics, see Nguyen et al. (2000) Chem Biol. 7 (7): 463-473; Spatola, A .; F. , In Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B .; Weinstein, eds. , Marcel Dekker, New York, p. 267 (1983). Spatola, A .; F. , Peptide Backbone Modifications (Review), Vega Data, Vol. 1, Issue 3, (March, 1983); Morley, Trends Pharm Sci (review), pp. 463-468 (1980); Hudson, D .; _{Al, Int J Pept Prot Res, 14} : 177-185 (1979) (- CH 2 NH -, CH 2 CH 2 -); Spatola et al, Life Sci, 38: 1243-1249 ( 1986) (- CH ₂ --S); Hann J. Chem. Soc. Perkin Trans. I, 307-314 (1982) (- CH-CH--, cis and trans); Almquist et al, J Med Chem, 23: 1392-1398 (1980) (- COCH 2 -); Jennings-White et al. , Tetrahedron Lett, 23: 2533 ( 1982) (- COCH 2 -); Szelke et al., European Appln. _{EP45665CA: 97: 39405 (1982)} (- CH (OH) CH 2 -); Holladay et al, Tetrahedron Lett, 24: 4401-4404 ( 1983) (- C (OH) CH 2 -); and Hruby , Life Sci, 31: 189-199 ( 1982) (- CH 2 --S--) see also; that each is incorporated herein by reference.

  It will also be apparent that any combination of natural amino acids and non-natural amino acid analogs can be used to produce the PCSB reagents described herein. Commonly found amino acid analogs that are not gene-encoded include, but are not limited to, ornithine (Orn); aminoisobutyric acid (Aib); benzothiophenylalanine (BtPhe); albidiyne (Abz); t-butylglycine (Tle); phenylglycine (PhG); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 1-naphthylalanine (1-Nal); 2-thienylalanine (2-Thi) 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); N-methylisoleucine (N-MeIle); homoarginine (Har); Nα-methylarginine (N-MeArg); phosphotyrosine (pTyr or pY); pipecolic acid (Pip) 4-chloro-phenylalanine (4-ClPhe); 4- fluorophenylalanine (4-FPhe); 1- aminocyclopropanecarboxylic acid (1-NCPC); and sarcosine (Sar). Any of the amino acids used in the PCSB reagent can be either D- or more usually the L-isomer.

Other non-naturally occurring analogs of amino acids that can be used to form the PCSB reagents described herein are biofunctional equivalents and are also useful in the compounds of the present invention. Peptoid and / or peptidomimetic compounds such as sulfonic acid analogs and boronic acid analogs of amino acids, including compounds having one or more amide bonds substituted by In the context of the present invention, for example, --CONH-- is defined as --CH ₂ NH--, so that radicals bonded by isosteres are held in the same orientation as radicals bonded by --CONH--. —, —NHCO——, —SO ₂ NH——, —CH ₂ O——, —CH ₂ CH ₂ ——, —CH ₂ S——, —CH ₂ SO——, — -CH - CH - (cis or _{trans), - COCH 2 -, -} CH (OH) CH 2 - and it can be substituted by 5-2 substituent (disubstituted) tetrazole. One or more residues of the PCSB reagents described herein can include peptoids.

  Thus, the reagent may also contain one or more N-substituted glycine residues (peptides having one or more N-substituted glycine residues may be referred to as “peptoids”). For example, in certain embodiments, one or more proline residues of any of the PCSB reagents described herein are replaced by N-substituted glycine residues. Specific N-substituted glycines that are preferred in this regard include, but are not limited to, N- (S)-(1-phenylethyl) glycine; N- (4-hydroxyphenyl) glycine; N- ( Cyclopropylmethyl) glycine; N- (isopropyl) glycine; N- (3,5-dimethoxybenzyl) glycine; and N-butylglycine. Other N-substituted glycines may also be suitable for substituting one or more amino acid residues within the PCSB reagent sequences described herein.

  The PCSB reagents described herein can be monomers, multimers, cyclized molecules, branched molecules, linkers, and the like. Any multimer (ie, dimer, trimer, etc.) or biofunctional equivalent thereof of the sequences described herein is contemplated. Multimers can be homomultimers, ie composed of identical monomers, eg, each monomer is the same peptide sequence. Alternatively, the multimer can be a heteromultimer, which means that not all monomers that make up the multimer are the same.

  Multimers can be used with or without monomers to each other or, for example, multi-antigenic peptides (MAPS) (eg, symmetrical MAPS), polymer backbones, eg, peptides and / or spacer units attached to a PEG backbone. It can be formed by direct binding to a substrate containing peptides connected in series.

Alternatively, linking groups can be added to the monomeric sequence and the monomers can be joined together to form multimers. Non-limiting examples of multimers that use linking groups include tandem repeats using a glycine linker; MAPS bound to a substrate via a linker and / or linear binding peptides bound to the backbone via a linker Including. The linking group may comprise the use of a bifunctional spacer unit (homobifunctional or heterobifunctional) as is known to those skilled in the art. By way of example and not limitation, using reagents such as succinimidyl-4- (p-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), succinimidyl-4- (p-maleimidophenyl) butyrate Many methods for including such spacer units in peptides that bind together are described in Pierce Immunotechnology Handbook (Pierce Chemical Co., Rockville, Ill.) And are available from Sigma Chemical Co. (St. Louis, Mo.) and Aldrich Chemical Co. (Milwaukee, Wis.) And is described in “Comprehensive Organic Transformations”, VCK-Verlagsgesellschaft, Weinheim / Germany (1989). An example of a linking group that can be used to link monomeric sequences together is --Y ₁ --F--Y ₂ , where Y ₁ and Y ₂ are the same or different and have 0 to 20 carbon atoms. , Preferably 0 to 8, more preferably 0 to 3 alkylene groups, and F is --O--, --S--, --S--S--,- C (O)-O--, --NR--, --C (O)-NR--, --NR--C (O)-O--, --NR--C ( One or more functional groups such as O)-NR--, --NR--C (S)-NR--, --NR--C (S)-O--. Y ₁ and Y ₂ can be optionally substituted by hydroxy, alkoxy, hydroxyalkyl, alkoxyalkyl, amino, carboxyl, carboxyalkyl, and the like. It should be understood that any suitable atom of the monomer can be attached to the linking group.

  Further, the PCSB reagents described herein can be linear, branched or cyclized. Monomer units can be cyclized or linked together to form multimers in a linear or branched fashion, in the form of rings (eg macrocycles), in the form of stars (dendrimers) or in the form of balls. (Eg fullerene). Those skilled in the art will readily recognize numerous polymers that can be formed from the monomeric sequences disclosed herein. In certain embodiments, the multimer is a cyclic dimer. Using the same terms as above, the dimer can be a homodimer or a heterodimer.

  Cyclic forms, whether monomers or multimers, are not limited to this, for example: (1) direct amide bond formation between nitrogen and C-terminal carbonyl, or for example with epsilon-aminocarboxylic acid Cyclization with an N-terminal amine and a C-terminal carboxylic acid, either by mediation of a spacer group, such as by condensation; (2) by forming an amide bond between, for example, an aspartic acid or glutamic acid side chain and a lysine side chain; Or cyclization by formation of a bond between the side chains of two residues by forming a disulfide bond between two cysteine side chains or between a penicillamine side chain and a cysteine side chain or between two penicillamine side chains (3) either side chain (eg aspartic acid or lysine) and either N-terminal amine or C-terminal carboxyl Cyclization by the formation of an amide bond between the respectively; and / or (4) binding of the two side chains by mediation of short carbon spacer group; can be produced by any of the bonds described above, such as.

  In addition, the PCSB reagents described herein can also include additional peptide or non-peptide components. Non-limiting examples of additional peptide components include spacer residues, such as two or more glycine (natural or derivatized) residues or aminohexanoic acid linkers on one or both ends or residues that can help solubilize peptide reagents. Contains an acidic residue (Asp or D) such as a group, for example aspartic acid. In certain embodiments, for example, the peptide reagent is synthesized as a multi-antigenic peptide (MAP). Typically, multiple copies (eg, 2-10 copies) of a peptide reagent are synthesized directly on a MAP carrier such as branched lysine or other MAP carrier core. See, for example, Wu et al., 2001 123 (28): 6778-84; Spetzler et al. (1995) Int J Pept Protein Res. 45 (1): 78-85.

  Non-limiting examples of non-peptide components (eg, chemical moieties) that can be included in the PCSB reagents described herein include one or more detectable labels, tags (eg, biotin, His-Tag, oligonucleotides), Dyes, members of binding pairs, etc. are included either at the end or within the peptide reagent. Non-peptide components can also be directly or via a spacer (eg, an amide group) (eg, one or more labels) at a position on a compound that is predicted to be non-interfering by quantitative structure activity data and / or molecular modeling. Via a covalent bond). The PCSB reagents described herein can also include a prion specific chemical moiety such as an amyloid specific dye (eg, Congo Red, Thioflavin, etc.). Derivatization of compounds (eg, labeling, cyclization, coupling chemical moieties, etc.) should not substantially interfere (and even improve) the binding properties, biological function and / or pharmacological activity of the reagent.

  The peptides described above can be prepared using standard methods known to those of skill in the art, including but not limited to expression from recombinant constructs and peptide synthesis.

(C. Examples of preferred peptides used as the basis for PCSB reagents)
Non-limiting examples of peptides useful for generating the pathogenic conformer specific binding reagents of the present invention are preferably derived from the sequences shown in Table 3. The peptides in the table are represented by the conventional one letter amino acid code and are shown with their amino terminus on the left and the carboxy terminus on the right. X indicates that any amino acid can be placed at that position.

  Table array

のいずれも、アミノおよび／またはカルボキシ末端にＧｌｙリンカー（Ｇｎ、ここでｎ＝１、２、３、または４）を場合により含み得る。角カッコ内のアミノ酸は、別のペプチドにおいてその位置で使用できる代わりの残基を示す。丸カッコは残基がペプチド試薬に存在してもよく、または存在しなくてもよいことを示す。「２」が続く二重丸カッコ（たとえば配列番号１１１）は、配列が二重カッコの間のペプチドのコピーを２個含むことを示す。コピー回数の表示に続く残基（たとえば配列番号１１１では「Ｋ」）は、二重カッコ間のペプチドの各コピーが伸長する残基を示す。それゆえ配列番号１１１は、そのカルボキシ末端がリジンのａ−およびｅ−アミノ官能基を介してリジン（Ｋ）残基にそれぞれ結合された、ＱＷＮＫＰＳＫＰＫＴＮペプチド配列（すなわち配列番号１４）のダイマーである。「ＭＡＰＳ」を含む配列は、多抗原性部位を備えたペプチドを示す。「分枝」という用語に先行する数字は、コピー数を示す。それゆえ配列番号１１２は、各末端にＧｌｙリンカーを備えた配列番号６７である、ＧＧＧＫＫＲＰＫＰＧＧＷＮＴＧＧＧを４コピー含有するが、配列番号１１３は、また各末端にＧｌｙリンカーを備えた配列番号６７である、ＧＧＧＫＫＲＰＫＰＧＧＷＮＴＧＧＧを８コピー含有する。

Can optionally include a Gly linker (Gn, where n = 1, 2, 3, or 4) at the amino and / or carboxy terminus. Amino acids in square brackets indicate alternative residues that can be used at that position in another peptide. The parentheses indicate that the residue may or may not be present in the peptide reagent. A double parenthesis followed by “2” (eg, SEQ ID NO: 111) indicates that the sequence contains two copies of the peptide between the double parentheses. Residues following the display of the number of copies (for example, “K” in SEQ ID NO: 111) indicate residues to which each copy of the peptide between double brackets extends. SEQ ID NO: 111 is therefore a dimer of the QWNKPSKPKTN peptide sequence (ie, SEQ ID NO: 14) whose carboxy terminus is attached to the lysine (K) residue via the lysine a- and e-amino functional groups, respectively. A sequence containing “MAPS” indicates a peptide with multiple antigenic sites. The number preceding the term “branch” indicates the copy number. Therefore SEQ ID NO: 112 contains 4 copies of GGGKKRPKPGGWNTGGGG, which is SEQ ID NO: 67 with Gly linker at each end, while SEQ ID NO: 113 is GGGGKKRPKPGGWNTGGGG, which is also SEQ ID NO: 67 with Gly linker at each end Containing 8 copies.

  In certain embodiments, peptide fragments are all of the name of “Prion-Specific Peptide Reagents”, each of which is hereby incorporated by reference in its entirety, of a co-pending patent application filed on August 13, 2004. US Patent Application No. 10 / 917,646, US Patent Application No. 11 / 056,950 filed on February 11, 2005, and International Application PCT / US2004 filed on August 13, 2004. Can be derived from any of the regions corresponding to residues 23-43 or 85-156, numbered according to the murine prion sequence shown in SEQ ID NO: 2 of / 026363 (eg 23-30, 86-111, 89 -112, 97-107, 113-135, and 136-156).

  In some embodiments, the peptide fragment is selected from any one of SEQ ID NOs: 14, 50, 51, 52, 12, 72, 68 or 115 to 219. In some embodiments, the peptide fragment is selected from any one of SEQ ID NOs: 14, 50, 51, 52, or 161-219. In some embodiments, the peptide fragment is selected from any one of SEQ ID NOs: 12, 72, 68 or 115-160. In some embodiments, the peptide fragment is selected from any one of SEQ ID NOs: 14, 50, or 68.

(D. Peptoid PCSB reagent)
In a particularly preferred embodiment, the PCSB reagent is a peptoid. Typically, the PCSB reagent is derived from a prion protein fragment. Preferred peptoids are described below.

(Peptoid design)
As a starting point, the peptoid PCSB reagent performs substitution of amino acid residues within the sequence of peptide fragments with N-substituted glycine, US Pat. Nos. 5,811,387; 5,831,005; 877,278; 5,977,301; 6,075,121; 6.251,433; and 6,033,631, as well as Simon et al. (1992 ) Proc. Natl. Acad. Sci. Synthesis of modified peptides using the methods described in USA 89: 9367 (these publications are incorporated herein by reference in their entirety), and pathogenic conformers by the methods described herein. Testing of modified peptides for binding to can be designed based on either the sequence of prion protein fragments described above or variants of such fragments. Further substitutions can be made according to the substitution scheme below until suitable reagents are realized.

In some embodiments, the PCSB reagent is
a) Providing a peptide fragment of the prion protein, wherein the first amino acid of the peptide fragment is N-substituted glycine,
i) Ala, Gly, Ile, Leu, Pro, and Val are substituted by N- (alkyl) glycine, N- (aralkyl) glycine, or N- (heteroarylalkyl) glycine;
ii) Asp, Asn, Cys, Gln, Glu, Met, Ser and Thr are N- (hydroxyalkyl) glycine, N- (alkoxy) glycine, N- (aminoalkyl) glycine, or N- (guanidinoalkyl) glycine Replaced by;
iii) Phe, Trp, and Tyr are substituted by N- (aralkyl) glycine, N- (heteroarylalkyl) glycine, N- (hydroxyaralkyl) glycine, or N- (alkoxyaralkyl) glycine;
iv) Arg, His, and Lys are substituted by a substitution scheme, substituted by N- (aminoalkyl) glycine, or N- (guanidinoalkyl) glycine;
b) replacing the second amino acid of the peptide fragment with an N-substituted glycine according to step a);
c) replacing the third amino acid of the peptide fragment with an N-substituted glycine according to step a);
d) optionally repeating step c) 1-27 times, thereby providing a designed peptoid PCSB reagent comprising 3-30 N-substituted glycines; and synthesizing the designed peptoid PCSB reagent; Designed by.

  Modified peptides can be tested for binding to pathogenic conformers according to the methods described herein. Further substitution of amino acid monomers with N-substituted glycines according to the above scheme can be performed and retested until a suitable bond is obtained (ie PCSB reagent that interacts preferentially with the pathogenic form of the prion).

  Methods for producing peptoids are disclosed in US Pat. Nos. 5,811,387 and 5,831,005, each of which is incorporated by reference in its entirety, as is the method disclosed herein. Incorporated herein by reference.

The pathogenic conformer specific binding reagent used in the method of the present invention is:
_{^{X a - (Q) n -X}} b
Can have the formula:
Each Q is independently an amino acid or an N-substituted glycine,-(Q) _n- defines a peptoid region;
X ^a is, _{H, (C} 1 -C ₆₎ alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, _{heterocycloalkyl, (C} 1 -C ₆₎ acyl, amino _{(C 1-6)} acyl An amino acid, an amino protecting group, or a polypeptide of 2 to about 100 amino acids, wherein X ^a is optionally substituted by a conjugation moiety optionally linked through a linker moiety;
X ^b is H, (C ₁ -C ₆ ) alkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, amino, alkylamino, dialkylamino, hydroxyl, (C ₁ -C ₆ ) alkoxy, aryl An oxy, aralkoxy, carboxy protecting group, amino acid, polypeptide of 2 to about 100 amino acids, wherein ^Xb is optionally substituted by a conjugation moiety optionally linked through a linker moiety;
n is from 3 to about 30 (ie, n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more);
Here, at least about 50% of the peptoid region-(Q) _n- includes, but is not limited to, N-substituted glycine.

  In some embodiments, each Q is independently an N-substituted glycine.

In some embodiments, PCSB ^reagents, X a - has the _(Q) wherein the n -X ^b, wherein, n, about 4 to about 30, preferably about 5 to about 30, wherein in peptoid region _- (Q) n - of at least about 50%, but are not limited to, include n-substituted glycine, however peptoid region _- (Q) n - are being limited thereto Not there:
(A) -AABA-;
(B) -AABAB-
(C) -ABACC-;
(D) -AAAAA-;
(E) -ABCBA-;
(F) -AABCA-; or (g) at least one subregion selected independently from -ABABA-; wherein A, B, and C are each a different N-substituted glycine. is there.

In some embodiments, X ^a is (C ₁ -C ₆ ) acyl or amino (C ₁ -C ₆ ), optionally substituted by a conjugating moiety, each optionally linked via a linker moiety. Acyl.

In some embodiments, each X ^a is (C ₁ -C ₆ ) acyl, each optionally substituted by a conjugating moiety selected from a bridging or binding reagent, each optionally linked via a linker moiety. or an amino _(C 1 -C ₆₎ acyl.

In some embodiments, X ^a is (C ₁ -C ₆ ) acyl or amino, each optionally substituted by a conjugation moiety selected from biotin or mercapto, optionally linked via a linker moiety. (C ₁ -C ₆ ) acyl.

In some embodiments, ^Xb is an amino acid optionally substituted by a conjugating moiety that is optionally linked through a linker moiety.

In some embodiments, X ^b is amino, alkylamino, dialkylamino.

In some embodiments, X ^b is amino.

  In some embodiments, n is about 5 to about 15; 5 to about 10; or 6.

  In some embodiments, n is 4-10, 4-8, 5-7, or 6.

In some embodiments, X ^b is an amino acid optionally substituted with a conjugating moiety and n is 6.

In some embodiments, the linker moiety contains a region having the formula — {NH (CH 2) _m C (O)} _p —.

  In some embodiments, m is 1-10.

  In some embodiments, m is 1-8.

  In some embodiments, m is 5.

  In some embodiments, p is 1-5.

  In some embodiments, p is 1-3.

  In some embodiments, p is 1 or 2.

In some embodiments, X ^b is an amino acid optionally substituted by a conjugating moiety that is optionally linked through a linker moiety, and n is 6.

In some embodiments, X ^b is amino, alkylamino, or dialkylamino; X ^a is H, (C ₁ -C ₆ ) alkyl, (C _1-6 ) acyl, amino (C _1- C ₆ ) an acyl, amino acid, or amino protecting group, wherein X ^a is optionally substituted by a conjugating moiety optionally attached through a linker moiety;

In some embodiments, X ^b is amino, alkylamino, or dialkylamino; X ^a is H, (C ₁ -C ₆ ) alkyl, (C _1-6 ) acyl, amino (C _1- C ₆ ) acyl, amino acid or amino protecting group, wherein X ^a is substituted by a conjugating moiety selected from a crosslinker or binder, optionally linked via a linker moiety; It is.

In some embodiments, X ^b is amino, alkylamino, or dialkylamino; X ^a is H, (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) acyl, amino (C _{1 -6)} acyl, an amino acid or an amino protecting group, wherein X ^a is coupled optionally through a linker moiety, is substituted by conjugated moiety comprising biotin or mercapto, at least a portion of the linker moiety formula _{- {NH (CH2) m C} (O)} p - having a; n is 6; m is an 1 to 10; p is 1-5.

In some embodiments, each Q is independently an amino acid or an N-substituted glycine having the formula — (NR—CH ₂ —CO) —, wherein each R is (C ₂ -C ₆ ) alkyl. , halo _(C 1 -C _{6) alkyl, (C} 2 -C _{6) alkenyl, (C} 2 -C _{6) alkynyl,} (C 6 _{-C 10)} cycloalkyl - aryl, amino _(C 1 -C ₆₎ alkyl , Ammonium (C ₁ -C ₆ ) alkyl, hydroxy (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy (C ₁ -C ₆ ) alkyl, carboxy, carboxy (C ₂ -C ₆ ) alkyl, carbamyl, carbamyl _(C 2 -C ₆₎ alkyl, guanidino, guanidino _(C 1 -C ₆₎ alkyl, amidino, amidino _(C 1 -C ₆₎ alkyl, _{thiol, (C} 1 -C ₆ ) Alkylthiol, of 2 to 10 carbon atoms alkylthioalkyl, N-containing heterocyclyl, N-containing heterocyclyl _(C 1 -C ₆₎ alkyl, imidazolyl, carbon atoms 4 to 10 imidazolylalkyl, piperidyl, 5 to 10 carbon atoms Independently selected from piperidylalkyl, indolyl, indolylalkyl of 9 to 15 carbon atoms, naphthyl, naphthylalkyl of 11 to 16 carbon atoms and aryl (C ₁ -C ₆ ) alkyl; wherein each R moiety Is optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxy and (C ₁ -C ₆ ) alkoxy.

In some embodiments, each Q is independently an amino acid or an N-substituted glycine having the formula — (NR—CH ₂ —CO) —, wherein each R is (C ₂ -C ₆ ) alkyl. , halo _(C 1 -C _{6) alkyl, (C} 2 -C _{6) alkenyl, (C} 2 -C _{6) alkynyl,} (C 6 _{-C 10)} cycloalkyl - aryl, amino _(C 1 -C ₆₎ alkyl , Hydroxy (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy (C ₁ -C ₆ ) alkyl, carboxy, carboxy (C ₂ -C ₆ ) alkyl, carbamyl, carbamyl (C ₂ -C ₆ ) alkyl, guanidino, guanidino _(C 1 -C ₆₎ alkyl, _{thiol, (C} 1 -C ₆₎ alkylthiol, of 2 to 10 carbon atoms alkylthioalkyl, imidazolyl, TansoHara 4 to 10 imidazolylalkyl, piperidyl, carbon atoms 5-10 piperidyl alkyl, indolyl, carbon atoms 9 to 15 amino indolylalkyl, naphthyl, carbon atoms 11 to 16 amino naphthylalkyl, diphenyl _{(C 1} - C ₆ ) alkyl or aryl (C ₁ -C ₆ ) alkyl independently selected; wherein each R moiety is independently selected from halogen, hydroxy and (C ₁ -C ₆ ) alkoxy 1-3 Optionally substituted by 1 substituent.

In some embodiments, each Q is independently an amino acid or an N-substituted glycine of the formula — (NR—CH ₂ —CO) —, wherein each R is halogen, hydroxy and (C ₁ -C ₆₎ is replaced by 1 to 3 substituents independently selected from _{alkoxy, (C} 2 -C ₆₎ alkyl, amino _(C 1 -C ₆₎ alkyl, hydroxy _(C 1 -C ₆₎ alkyl , (C ₁ -C ₆ ) alkoxy (C ₁ -C ₆ ) alkyl, guanidino (C ₁ -C ₆ ) alkyl, indolylalkyl having 9 to 15 carbon atoms, naphthylalkyl having 11 to 16 carbon atoms, diphenyl Independently selected from (C ₁ -C ₆ ) alkyl or aryl (C ₁ -C ₆ ) alkyl.

  In some embodiments, each Q is independently an amino acid or N- (4-aminobutyl) glycine, N- (1-phenylethyl) glycine, N- (2-aminoethyl) glycine, N- (2- [4-methoxyphenyl] ethyl) glycine, N- (2-methoxyethyl) glycine, N- (2-hydroxyethyl) glycine, N-((1H-indol-3-yl) methyl) glycine Or an N-substituted glycine selected from N-benzylglycine.

  In some embodiments, each Q is independently an amino acid or an N-substituted glycine selected from N- (4-aminobutyl) glycine or N-benzylglycine.

  In some embodiments, each Q is independently an N-substituted glycine.

In some embodiments, the peptoid region-(Q) _n -includes, but is not limited to, at least 3 or at least 4 N-substituted glycines charged at physiologically relevant pH. including. In some embodiments, the charge is positive. In some embodiments, the remaining N-substituted glycine in the peptoid region is neutral at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -is, but is not limited to, 2-6, 3-5, charged at physiologically relevant pH, Or four N-substituted glycines. In some embodiments, the charge is positive. In some embodiments, the remaining N-substituted glycine in the peptoid region is neutral at physiologically relevant pH.

In some embodiments, the two N-substituted glycine residues of the peptoid region-(Q) _n- are positively charged at physiologically relevant pH, and the remaining N-substituted glycine residues of the peptoid region are Neutral at physiologically relevant pH.

In some embodiments, the three N-substituted glycine residues in the peptoid region-(Q) _n- are positively charged at physiologically relevant pH, and the remaining N-substituted glycine residues in the peptoid region are Neutral at physiologically relevant pH.

In some embodiments, the four N-substituted glycine residues in the peptoid region-(Q) _n- are positively charged at physiologically relevant pH, and the remaining N-substituted glycine residues in the peptoid region are Neutral at physiologically relevant pH.

In some embodiments, the 5 N-substituted glycine residues of the peptoid region-(Q) _n- are positively charged at physiologically relevant pH, and the remaining N-substituted glycine residues of the peptoid region are Neutral at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -is polyionic at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -is polycationic at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -is polyanionic at a physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -has a net charge of at least 3+ at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -has a net charge of at least 4+ at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -has a net charge of 2+ to 6+ at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -has a net charge of 3+ to 5+ at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -has a net charge of 4+ at physiologically relevant pH.

In some embodiments, the peptoid region-(Q) _n -includes, but is not limited to, at least 3 N-substituted glycines that are positively charged at physiologically relevant pH. .

In some embodiments, the peptoid region-(Q) _n -includes, but is not limited to, at least 4 N-substituted glycines that are positively charged at physiologically relevant pH. .

In some embodiments, the peptoid region-(Q) _n -includes, but is not limited to, 2-6 N-substituted glycines that are positively charged at physiologically relevant pH. Including.

In some embodiments, the peptoid region-(Q) _n -includes, but is not limited to, 3-5 N-substituted glycines that are positively charged at physiologically relevant pH. Including.

In some embodiments, the peptoid region-(Q) _n -includes, but is not limited to, four N-substituted glycines that are positively charged at physiologically relevant pH.

In some embodiments, the peptoid region - _{(Q) n} - of the N-substituted glycine of the formula _{- (NR-CH 2 -CO)} - have, in the formula, _{R, (C} 2 -C ₆₎ alkyl, Halo (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, (C ₆ -C ₁₀ ) cycloalkyl-aryl, amino (C ₁ -C ₆ ) alkyl, Ammonium (C ₁ -C ₆ ) alkyl, hydroxy (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy (C ₁ -C ₆ ) alkyl, carboxy, carboxy (C ₂ -C ₆ ) alkyl, carbamyl , carbamyl _(C 2 -C ₆₎ alkyl, guanidino, guanidino _(C 1 -C ₆₎ alkyl, amidino, amidino _(C 1 -C ₆₎ alkyl, _{thiol, (C} 1 -C ₆₎ alkyl Ruthiol, alkylthioalkyl having 2 to 10 carbon atoms, N-containing heterocyclyl, N-containing heterocyclyl (C ₁ -C ₆ ) alkyl, imidazolyl, imidazolylalkyl having 4 to 10 carbon atoms, piperidyl, piperidyl having 5 to 10 carbon atoms Independently selected from alkyl, indolyl, indolylalkyl of 9 to 15 carbon atoms, naphthyl, naphthylalkyl of 11 to 16 carbon atoms, and aryl (C ₁ -C ₆ ) alkyl; wherein each R moiety is Optionally substituted by 1 to 3 substituents independently selected from halogen, hydroxy and (C ₁ -C ₆ ) alkoxy, the peptoid region-(Q) _n -is not limited thereto No but at least 3, at least 4, 2-6, 3-5, or 4 It includes substituted glycine, where R is a moiety that is charged at physiologically relevant pH.

  In some embodiments, all N-substituted glycines in the peptoid region are adjacent.

  In some embodiments, the peptoid PCSB reagent includes, but is not limited to, at least one conjugating moiety.

  In some embodiments, the peptoid PCSB reagent includes, but is not limited to, at least one conjugation moiety linked via a linker moiety.

In a preferred embodiment, the peptoid PCSB reagent includes, but is not limited to, an amino terminal region, a carboxy terminal region, and at least one peptoid region between the amino terminal region and the carboxy terminal region, wherein The peptoid region includes, but is not limited to, about 3 to about 30 N-substituted glycines and optionally one or more amino acids. In some embodiments, the peptoid region includes, but is not limited to, about 4 to about 30, or about 5 to about 30 N-substituted glycines. In some embodiments, the peptoid region includes, but is not limited to, about 4 to about 30, or about 5 to about 30 N-substituted glycines and:
(A) -AABA-;
(B) -AABAB-
(C) -ABACC-;
(D) -AAAAA-;
(E) -ABCBA-;
(F) -AABCA-; or (g) comprising a peptoid subregion selected from -ABABA-;
Here, A, B, and C are different N-substituted glycines, and each sub-region is read from left to right from the amino terminus toward the carboxy terminus.

  In some embodiments, the peptoid region comprises, but is not limited to, about 50 to about 100%, about 75 to about 100%, or 100% N-substituted glycine.

  In some embodiments, the peptoid region is about 5 to about 50, about 5 to about 30, about 5 to about 15, about 5 to about 7, or 6 in length. It is a subunit.

  In some embodiments, the peptoid reagent has a total length of about 5 to about 50, about 5 to about 30, about 5 to about 15, or about 6 to about 9 subunits.

  In some embodiments, the at least one peptoid region is greater than about 50%, greater than about 75%, or greater than about 90% of the total length of the peptoid reagent.

  In some embodiments, all N-substituted glycines are adjacent within the peptoid region.

In some embodiments, the N-substituted glycine of the peptoid region has the formula — (NR—CH ₂ —CO) —, wherein R is as defined throughout the specification.

  In some embodiments, the peptoid region is polyionic at physiologically relevant pHs and has features according to any of the embodiments described throughout this specification for charged peptoid regions.

  In other embodiments, the PCSB reagent comprises a peptoid region having 3-15 contiguous N-substituted glycines, wherein the peptoid region has a net charge at a physiologically relevant pH. In some embodiments, the net charge is a net positive charge, such as a net charge of at least 3+ or at least 4+ at physiologically relevant pH. In some embodiments, the reagent itself has a net charge of 2 + -6 +, 3 + -5 +, or 4+ at physiologically relevant pH.

  In some embodiments, at least two, at least three, or at least four of the adjacent N-substituted glycines in the peptoid region are charged at a physiologically relevant pH. In further embodiments, at least two of the adjacent N-substituted glycines in the peptoid region are at least selected from primary amino, secondary amino, tertiary amino, ammonium (quaternary amino), guanidino, amidino, or N-containing heterocyclyl. Contains one part.

  In still further embodiments, at least two of the adjacent N-substituted glycines in the peptoid region are selected from, but not limited to, primary amino, secondary amino, ammonium, guanidino, amidino, or N-containing heterocyclyl. At least one N substituent.

  In yet further embodiments, at least two of the adjacent N-substituted glycines contain an N substituent that is an R group according to the definitions provided herein.

  In still further embodiments, the peptoid PCSB reagent includes, but is not limited to, the peptoid region of six adjacent N-substituted glycines, and the peptoid PCSB reagent itself is 3+ or at physiologically relevant pH. It has a net charge of 4+.

A “peptoid reagent” is a molecule having an amino terminal region, a carboxy terminal region, and at least one “peptoid region” between the amino terminal region and the carboxy terminal region. The amino terminal region refers to the region on the amino terminal side of the reagent that typically does not contain any N-substituted glycines. The amino terminal region can be H, alkyl, substituted alkyl, acyl, amino protecting group, amino acid, peptide, and the like. In some embodiments, the amino-terminal region corresponds to X ^a. The carboxy-terminal region refers to the carboxy-terminal region of the peptoid that does not contain any N-substituted glycines. The carboxy terminal region can include H, alkyl, alkoxy, amino, alkylamino, dialkylamino, carboxy protecting groups, amino acids, peptides, and the like. In some embodiments, the carboxy terminal region corresponds to ^Xb . In some embodiments, the peptoid PCSB reagent has about 5 to about 50 subunits; about 5 to about 30 subunits; about 5 to about 15 subunits; or about 6 to about 9 subunits. It has the full length of the subunit. In some embodiments, the peptoid is a carboxy terminal amide. The peptoid region generally refers to the portion of the PCSB reagent in which at least three of the amino acids are replaced by N-substituted glycines.

The “peptoid region” (also referred to herein as “-(Q) _n- ”) starts with the N-substituted glycine closest to the amino terminus and includes that N-substituted glycine and is the closest to the carboxy terminus. Can be identified as the region containing that N-substituted glycine. In some embodiments, the peptoid region is, but is not limited to, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%. , At least about 95%, at least about 99%, or 100% N-substituted glycine. In some embodiments, the peptoid region comprises, but is not limited to, about 25 to about 100%; about 50 to about 100%; about 75 to about 100% N-substituted glycine. In some embodiments, the peptoid region includes, but is not limited to, 100% N-substituted glycine. In some embodiments, the peptoid region is greater than about 50% (eg, about 50-100%) of the total length of the peptoid PCSB reagent. In some embodiments, the peptoid region is greater than about 60% (eg, about 60-100%) of the total length of the peptoid reagent. In some embodiments, the peptoid region is greater than about 75% (eg, about 75-100%) of the total length of the peptoid reagent. In some embodiments, the peptoid region is greater than about 90% (eg, about 90-100%) of the total length of the peptoid reagent. In some embodiments, the peptoid region is 100% of the total length of the reagent.

  In some embodiments, the peptoid region contains at least 3 N-substituted glycines. In some embodiments, the peptoid region contains at least 4 N-substituted glycines. In some embodiments, the peptoid region contains at least 5 N-substituted glycines. In some embodiments, the peptoid region contains at least 6 N-substituted glycines. In some embodiments, the peptoid region contains 3 to about 30; about 5 to about 30 N-substituted glycines; and optionally one or more amino acids. In some embodiments, the peptoid region has about 5 to about 50 subunits, 5 to about 30, 5 to about 15, 5 to about 10, 5 to about 9, subunits, 5 to about 8 or 5 to about 7 lengths. In some embodiments, the peptoid region is about 3, 4, 5, 6, 7, 8, 9, or 10 subunits long. In some embodiments, the peptoid region is 6 subunits long. In some embodiments, all of the N-substituted glycines in the peptoid region are in close proximity. In some embodiments, all of the subunits of the peptoid region are N-substituted glycines.

  In further embodiments, the PCSB reagent is, but is not limited to, 4-12, 4-10, 4-9, 4-8, 5-7, or Contains the peptoid region of six adjacent N-substituted glycines.

  According to some embodiments, the peptoid region can be polyionic at physiologically relevant pH. The term “polyionic” means that the peptoid region contains two or more residues that are charged at physiologically relevant pH. In some embodiments, the peptoid region is polycationic or polyanionic at physiologically relevant pH. In further embodiments, the peptoid region has a net charge of at least 3+ or at least 4+ at physiologically relevant pH. In yet further embodiments, the peptoid region has a net charge of 2 + -6 +, 3 + -5 +, or 4+ at physiologically relevant pH.

  Non-limiting examples of charged N-substituted glycine residues include N- (5-aminopentyl) glycine, N- (4-aminobutyl) glycine, N- (3-aminopropyl) glycine, N- (2 -Aminoethyl) glycine, N- (5-guanidinopentyl) glycine, N- (4-guanidinobutyl) glycine, N- (3-guanidinopropyl) glycine, and N- (2-guanidinoethyl) glycine.

  In some embodiments, the peptoid region contains at least 3 or at least 4 N-substituted glycines that are positively charged at physiologically relevant pH.

  In some embodiments, the peptoid region contains 2-6, 3-5, or 4 amino N-substituted glycines that are positively charged at physiologically relevant pH.

In some embodiments, the peptoid region contains residues having the formula — (NR—CH ₂ —CO) —, wherein at least 3, at least 4, 2-6, 3 of residues ~ 5, or 4, are charged at a physiologically relevant pH.

In some embodiments, the charged residue of the peptoid region has the formula — (NR—CH ₂ —CO) —, wherein R is amino (C ₁ -C ₆ ) alkyl, ammonium ( Independent of C ₁ -C ₆ ) alkyl, guanidino, guanidino (C ₁ -C ₆ ) alkyl, amidino, amidino (C ₁ -C ₆ ) alkyl, N-containing heterocyclyl and N-containing heterocyclyl (C ₁ -C ₆ ) alkyl Wherein each R moiety is optionally substituted with 1 to 3 substituents independently selected from halogen, C ₁ -C ₃ methoxy, and C ₁ -C ₃ alkyl. In some embodiments, R is amino (C ₁ -C ₆ ) alkyl, such as aminobutyl.

  In some embodiments, the PCSB reagent has a net charge of at least 3+ or at least 4+ at physiologically relevant pH. In yet further embodiments, the reagent has a net charge of 2 + -6 +, 3 + -5 +, or 4+ at physiologically relevant pH.

The peptoid region of the PCSB reagent used in the method of the present invention is a sequence of consecutive N-substituted glycines of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more residues. May contain at least one peptoid subregion. In some embodiments, the peptoid region is:
(A) -AABA-;
(B) -AABAB-
(C) -ABACC-;
(D) -AAAAA-;
(E) -ABCBA-;
(F) -AABCA-; or (g) -ABABA-;
Containing at least one peptoid subregion selected independently from
A, B, and C each represent a different N-substituted glycine. For example, each A occurring in a sub-region refers to a specific N-substituted glycine and each B occurring in a sub-region refers to another specific N-substituted glycine, but A and B are different from each other. Thus, C is an N-substituted glycine that is different from either A or B. Subregion sequence means read from left to right in amino to carboxy orientation. In some embodiments, when A is a hydrophobic residue, B is a hydrophilic residue and vice versa. In some embodiments, the peptoid subregions are homologous, ie contain only one type of N-substituted glycine. In some embodiments, when A is an aliphatic residue, B is a cyclic residue. In some embodiments, when B is an aliphatic residue, A is a cyclic residue. In some embodiments, both A and B are aliphatic. In some embodiments, A and B are aliphatic and C is cyclic. In some embodiments, all N-substituted glycines are aliphatic (eg,-(N- (2-methoxyethyl) glycine) ₂ -N- (4-aminobutyl) glycine, for example, in sub-region -AABA- -(N- (2-methoxyethyl) glycine)-, where A is N- (2-methoxyethyl) glycine and B is N- (4-aminobutyl) glycine).

In some embodiments, the peptoid region contains tripeptoids, ie three adjacent N-substituted glycines. Exemplary tripeptoid peptoid subregions are-(N- (2- (4-hydroxyphenyl) ethyl) glycine) ₂ -N- (4-guanidinobutyl) glycine-, -N- (4- Aminobutyl) glycine- (V) _2- , where V is N-benzylglycine or N- (2-methoxyethyl) glycine, -N-benzylglycine-WN-benzylglycine-, where And W is N- (4-aminobutyl) glycine or N- (2-methoxyethyl) glycine, and -N- (4-aminoethyl) glycine- (N- (2- (4-methoxyphenyl) ethyl) Glycine) _2- . In some embodiments, the tripeptoid subregion contains at least one aliphatic residue and at least one cyclic residue (eg, (A) ₂ -B, B ₂ -A, or B-A-B, where A is an aliphatic residue and B is a cyclic residue).

  In some embodiments, the peptoid subregion is a dipeptoid, such as N- (4-aminobutyl) glycine- (S) -N- (1-phenylethyl) glycine dipeptoid.

  PCSB reagents useful in the methods of the present invention include monomers, multimers, cyclized molecules, branched molecules, linkers, and the like. Any multimer (ie, dimer, trimer, etc.) or biofunctional equivalent thereof of the sequences described herein is contemplated. Multimers can be homomultimers, i.e. composed of identical monomers. Instead, the multimer can be a heteromultimer, i.e. not all monomers that make up the multimer are the same.

  Multimers can be used with or without monomers to each other or, for example, multi-antigenic peptides (MAPS) (eg, symmetrical MAPS), polymer backbones, eg, peptides and / or spacer units attached to a PEG backbone. It can be formed by direct binding to a substrate containing peptides connected in series. Alternatively, linkers can be added to the monomers and joined together to form multimers. Non-limiting examples of multimers using linkers include, for example, tandem repeats using glycine linkers, MAPS linked to substrates via linkers and / or linear binding peptides linked to the backbone via linkers including. The linker moiety may include the use of bifunctional spacer units (either homobifunctional or heterobifunctional) as are known to those skilled in the art.

  In some embodiments, the PCSB reagent is at least about 2-fold; 5-fold; 10-fold; 20-fold; 50-fold; 100-fold; 200-fold; 500 than the affinity of the non-pathogenic form of the conformational disease protein. Interacts with pathogenic conformers with a fold; or 1000-fold greater affinity. In some embodiments, the affinity is at least about 10 times greater than the affinity of the non-pathogenic form of the conformational disease protein. In some embodiments, the affinity is at least 100 times greater.

Any of the PCSB reagents described herein can be used in the detection method of the present invention. In some embodiments, the detection method of the present invention comprises:
_{^{X a - (Q) n -X}} b
Using a PCSB reagent having the formula:
Each Q is independently an amino acid or an N-substituted glycine,-(Q) _n- defines a peptoid region;
X ^a is, _{H, (C} 1 -C ₆₎ alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, _{heterocycloalkyl, (C} 1 -C ₆₎ acyl, amino _{(C 1-6)} acyl An amino acid, an amino protecting group, or a polypeptide of 2 to about 100 amino acids, wherein X ^a is optionally substituted by a conjugation moiety optionally linked through a linker moiety;
X ^b is H, (C ₁ -C ₆ ) alkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, amino, alkylamino, dialkylamino, hydroxyl, (C ₁ -C ₆ ) alkoxy, aryl An oxy, aralkoxy, carboxy protecting group, amino acid, polypeptide of 2 to about 100 amino acids, wherein ^Xb is optionally substituted by a conjugation moiety optionally linked through a linker moiety;
n is from 3 to about 30; where at least about 50% of the peptoid region-(Q) _n- includes, but is not limited to, N-substituted glycines.

In some such embodiments, n is from about 4 to about 30, preferably from about 5 to about 30, and the peptoid region-(Q) _n -is:
(A) -AABA-;
(B) -AABAB-
(C) -ABACC-;
(D) -AAAAA-;
(E) -ABCBA-;
(F) -AABCA-; or (g) -ABABA-;
Wherein A, B, and C are each different N-substituted glycines.

In some embodiments of the detection method, the PCSB reagent contains an amino terminal region, a carboxy terminal region, and at least one peptoid region between the amino terminal region and the carboxy terminal region, wherein the peptoid region is Contains from about 3 to about 30 N-substituted glycines and optionally one or more amino acids. In some such embodiments, the peptoid region is:
(A) -AABA-;
(B) -AABAB-
(C) -ABACC-;
(D) -AAAAA-;
(E) -ABCBA-;
(F) -AABCA-; and (g) -ABABA-;
Wherein A, B, and C are each different N-substituted glycines.

In some embodiments of the methods of the invention, the PCSB reagent includes, but is not limited to, a peptoid analog of a 3-30 amino acid peptide fragment of a prion protein, wherein the peptide fragment is SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 77,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, or 228, where:
(A) at least one non-proline residue of the peptide fragment is substituted with an N-substituted glycine to form a peptoid analog; or (b) at least 5 amino acid residues of the peptide fragment are each with an N-substituted glycine. Is substituted to form a peptoid analog.

In some embodiments of the above method, replacement of any one or more amino acid residues of a peptide fragment with an N-substituted glycine corresponds to the following substitution scheme:
i) Ala, Gly, Ile, Leu, Pro, and Val are substituted by N- (alkyl) glycine, N- (aralkyl) glycine, or N- (heteroarylalkyl) glycine;
ii) Asp, Asn, Cys, Gln, Glu, Met, Ser, and Thr are N- (hydroxyalkyl) glycine, N- (alkoxy) glycine, N- (aminoalkyl) glycine, or N- (guanidinoalkyl) Replaced by glycine;
iii) Phe, Trp, and Tyr are substituted by N- (aralkyl) glycine, N- (heteroarylalkyl) glycine, N- (hydroxyaralkyl) glycine, or N- (alkoxyaralkyl) glycine;
iv) Arg, His, and Lys are replaced by N- (aminoalkyl) glycine or N- (guanidinoalkyl) glycine.

  In some such embodiments, the PCSB reagent is a peptoid analog of the 5-30 amino acid peptide fragment of the prion protein described above.

(Preferable peptoid region sequence)
Table 4 shows exemplary peptoid regions (amino to carboxy orientation) suitable for preparing PCSB reagents for use in the present invention. Table 5 shows the abbreviated keys used in Table 1. Table 6 gives the related structure for each sequence.

具体的なＰＣＳＢ試薬の調製は以下で説明する。

Specific PCSB reagent preparation is described below.

  In some embodiments of the methods of the invention, the PCSB reagent is, but is not limited to, a sequence as described herein, eg, SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, or 241 sequences. In some embodiments, the PCSB reagent comprises a sequence selected from SEQ ID NOs: 229, 230, 232, 233, 234, 235, 237, 238, 239, or 240. In some embodiments, the PCSB reagent comprises a sequence selected from, but not limited to, SEQ ID NOs: 229, 230, 235, 237, 238, 239, or 240. In some embodiments, the PCSB reagent comprises a sequence selected from, but not limited to, SEQ ID NOs: 230, 237, 238, 239, or 240. In some embodiments, the PCSB reagent used in the method comprises, but is not limited to, a sequence selected from SEQ ID NOs: 229, 236, 231, 232, 233, 234 or 235. In some embodiments, the PCSB reagent comprises a sequence selected from, but not limited to, SEQ ID NOs: 230, 237, 238, 239, or 240. In some such embodiments, the PCSB reagent comprises, but is not limited to, SEQ ID NO: 230, 237 or 240. In some such embodiments, the PCSB reagent comprises, but is not limited to, SEQ ID NO: 240.

(E. Examples of preferred peptoids)
In a preferred embodiment, the pathogenicity specific conformer binding reagent used in the method of the invention is a reagent that interacts preferentially with the pathogenic conformer of the prion protein, eg as described in Example 4. One or more of the reagents indicated by I, II, VII, IX, X, XIa, XIb, XIIa, or XIIb.

In a preferred embodiment, the PCSB reagent comprises human prion protein fragments PrP _19-30 (SEQ ID NO: 242), PrP _23-30 (SEQ ID NO: 243), PrP _100-111 (SEQ ID NO: 244), PrP _101-110 (SEQ ID NO: _{245), PrP 154-165} (SEQ ID NO: _{246), PrP 226-237} (SEQ ID NO: 247) peptide, SEQ ID NO: 14, a peptoid derived from SEQ ID NO: 50 or SEQ ID NO: 68,.

  In a particularly preferred embodiment, the PCSB reagent is

の構造を含む。

Including the structure.

(F. Identification of PCSB reagent used in the method of the present invention)
The PCSB reagent used in the method of the present invention interacts preferentially with the pathogenic conformer of the prion protein. This property is due to any known binding assay, for example standard immunoassays such as ELISA, Western blots; labeled peptides; ELISA-like assays; and / or cell-based assays, in particular “by binding of pathogenic conformers to PCSB reagents It can be tested using the assay described in the following section with respect to “detection of pathogenic conformers”.

  One convenient way to test the specificity of the PCSB reagent used in the method of the invention is to select a sample containing both pathogenic and non-pathogenic conformers. Typically, such samples include tissue from affected animals. PCSB reagents as described herein that are known to specifically bind to pathogenic forms are bound to a solid support (by methods well known in the art and further described below), It is used to separate ("pull down") pathogenic conformers from other sample components to obtain quantitative values that are directly related to the number of reagent-protein binding interactions on the solid support. This result can be compared with the results of PCSB reagents with unknown binding specificity to determine whether such reagents can preferentially interact with pathogenic conformers.

  These assays may take advantage of the fact that pathogenic conformers are generally resistant to certain proteases, such as proteinase K. The same protease can degrade non-pathogenic conformers of conformational disease proteins. Thus, when using protease, the sample can be divided into two equal parts. Protease can be added to the second sample to perform the same test. Any reagent-protein binding interaction in the second sample can be attributed to the pathogenic conformer because any non-pathogenic conformer is degraded by the protease in the second sample.

(IV. Detection of pathogenic conformers by binding of pathogenic conformers to PCSB reagent)
The described PCSB reagents can be used to screen samples (eg, biological samples such as blood, brain, spinal cord, CSF or organ samples), eg, the presence of pathogenic forms of conformational disease proteins in these samples or It can be used in a wide variety of assays to detect the absence. Unlike many current diagnostic reagents, the PCSB reagents described herein are substantially any type of biological or non-biological, including blood samples, blood products, CSF, or biopsy samples. Detection with a sample is also possible. Detection methods can be used, for example, in methods of diagnosing conformational protein diseases and in any other situation where it is important to know the presence or absence of pathogenic conformers.

(Use of pathogenic conformer specific binding reagents as either capture or detection reagents)
PCSB reagents used in the methods of the present invention are typically derived from prion protein fragments and preferentially interact with pathogenic conformers of prion proteins. It is expected that at least some of these PCSB reagents will interact preferentially to the same extent with both pathogenic prions and other pathogenic conformers. In samples expected to contain pathogenic conformers of more than one conformational disease protein, or determining the type of pathogenic conformers present is important for the purpose of the method In some cases, pathogenic conformer specific binding reagents should be used for detection in combination with CDPSB reagents having different binding specificities and / or affinities for different types of conformational disease proteins. For example, if a pathogenic conformer specific binding reagent is used as a capture reagent, a conformational disease protein specific binding reagent should be used as a detection reagent, and vice versa. However, if the particular sample being assayed is expected to contain only one pathogenic conformer, or determining that a pathogenic conformer is present is not critical to the purpose of the method In this case, the PCSB reagent can now be used as both a capture and detection reagent.

  Some PCSB reagents used in the methods of the present invention are compared to other pathogenic conformers so that the detection assay can be performed in a manner that allows detection of only non-prion pathogenic conformers. It interacts preferentially to a different extent with pathogenic prions. In this case, the PCSB reagent can be used as both a capture and detection reagent.

(Method of using pathogenic conformer specific binding reagent as capture agent)
In a preferred embodiment, the present invention provides a pathogenic conformer-specific binding reagent in a sample suspected of containing a non-prion pathogenic conformer and, if present, a pathogenic conformer. By contacting under conditions that allow binding of the reagent; and if there is a pathogenic conformer in the sample, detecting the presence of the pathogenic conformer by its binding to the reagent; Provided is a method for detecting the presence of a non-prion pathogenic conformer in which a pathogenic conformer-specific binding reagent is derived from a prion protein fragment and the pathogenic conformer of a prion protein Interact with preferentially.

  For use in the method of the present invention, the sample may be anything known or suspected of containing non-prion pathogenic conformers. The sample can be a biological sample (ie, a sample prepared from a living or formerly living organism) or a non-biological sample. Suitable biological samples include, but are not limited to, organs, whole blood, blood fractions, blood components, plasma, platelets, serum, cerebrospinal fluid (CSF), brain tissue, nervous system tissue, muscle Includes tissue, bone marrow, urine, tears, non-neural tissues, organs, and / or biopsy or autopsy. Preferred biological samples include plasma and CSF.

  The sample is one or more PCSB reagents described herein under conditions that permit binding of the PCSB reagent to the pathogenic conformer if the pathogenic conformer is present in the sample. Contact with. Determining specific conditions based on the disclosure herein is well within the ability of those skilled in the art. Typically, the sample and PCSB reagent are placed in a suitable buffer (eg, TBS buffer at pH 7.5) at about neutral pH at a suitable temperature (eg, about 4 ° C.) for a suitable period (eg, about 1 hour). Incubate together (˜overnight) to cause binding.

  In such embodiments of the method, the pathogenic conformer specific binding reagent is a capture reagent and the presence of the pathogenic conformer in the sample is associated with the pathogenic conformer specific binding reagent. Detected by its binding. After capture, the presence of a pathogenic conformer can be detected by the exact same pathogenic conformer specific binding reagent that acts simultaneously as a capture and detection reagent. Instead, there may be a separate detection reagent, which may be either a different pathogenic conformer specific binding reagent, or preferably one or more conformation disease protein specific binding reagents. In a preferred embodiment, after the capture step, unbound sample is removed and the pathogenic conformer is dissociated from the complex it has formed with the capture reagent and denatured for detection. The capture reagent is preferably bound to a solid support.

(Method of using pathogenic conformer specific binding reagent as detection reagent)
In other embodiments, the invention provides conformational disease protein-specific binding to a sample suspected of containing a non-prion pathogenic conformer that binds to both pathogenic and non-pathogenic forms of the conformational disease protein. Contacting a binding agent, if present, under conditions that allow binding of the CDPSB reagent to the conformational disease protein to form a first complex; and a PCSB reagent in the first complex; If there is a pathogenic conformer in the sample, contacting under conditions that allow binding and detecting the presence of the pathogenic conformer by its binding to the PCSB reagent, Of non-prion pathogenic conformers, wherein the PCSB reagent is derived from a prion protein fragment and pathogenic prionta Park proteins preferentially interact.

(A. Reagent for capturing pathogenic conformers)
In a preferred embodiment, the capture reagent is a pathogenic conformer specific binding reagent that is derived from a prion protein fragment and interacts preferentially with the pathogenic conformer of the prion protein. In other embodiments, the capture reagent is a conformational disease protein-specific binding reagent that binds both pathogenic and non-pathogenic forms of the conformational disease protein.

  The capture reagent contacts the sample under conditions that allow any non-prion pathogenic conformer in the sample to bind to the reagent to form a complex. Such binding conditions are readily determined by those skilled in the art and are further described herein. Typically, the method is performed in the wells of a microtiter plate or in a small volume plastic tube, but any convenient container is suitable. The sample is generally a liquid sample or suspension and can be added to the reaction vessel before or after the capture reagent.

  The capture reagent is preferably bound to a solid support described in more detail in the following section. In some embodiments, the solid support is bound prior to application of the sample. A solid support (eg, magnetic beads) is first reacted with a capture reagent as described herein such that the capture reagent is sufficiently immobilized on the support. The solid support to which the capture reagent is bound is then contacted with a sample suspected of containing the pathogenic conformer under conditions that allow binding of the capture reagent to the pathogenic conformer.

  Instead, the capture reagent is first contacted with a sample suspected of containing a non-prion pathogenic conformer prior to binding to the solid support, followed by binding of the capture reagent to the solid support. (Eg, the reagent can be biotinylated and the solid support can include avidin or streptavidin).

  In certain embodiments, after a complex is formed between the capture reagent and the pathogenic conformer, it binds to the capture reagent, including unbound sample material (ie, any unbound pathogenic conformer). Any component of the sample that has not been removed) can be removed. For example, if the capture reagent is bound to a solid support, unbound material can be reduced by separating the solid support from the reaction solution (containing unbound sample material), eg, by centrifugation, precipitation, filtration, magnetic force, etc. Can do. Prior to performing the next step of the method, the solid support with the complex may optionally be subjected to one or more washing steps to remove any residual sample material.

  In some embodiments, after removal of unbound sample material and optional washing, the bound pathogenic conformer is dissociated from the complex and detected using any known detection method. Instead, the bound pathogenic conformer in the complex is detected without dissociation from the capture reagent.

(B. Dissociation and denaturation of pathogenic conformers)
After binding to the capture reagent to form a complex, the pathogenic conformer can be processed to facilitate detection of the pathogenic conformer.

  In some embodiments, unbound material is removed and then the pathogenic conformer is dissociated from the complex. “Dissociation” refers to the physical separation of a pathogenic conformer from a capture reagent that allows the pathogenic conformer to be detected separately from the capture reagent. Dissociation from the complex of pathogenic conformers can be achieved using, for example, low concentrations (eg, 0.4-1.0 M) of guanidinium hydrochloride or guanidinium isothiocyanate.

  If the CDPSB reagent used in the method can detect only denatured protein, the dissociated pathogenic conformer is also denatured. “Denature” refers to the destruction of the native conformation of a polypeptide. For example, if the reagent contains an activatable reactive group (eg, a photoreactive group) that covalently bonds the reagent and the pathogenic conformer, denaturation can be achieved without dissociating from the reagent.

  In a preferred embodiment, pathogenic conformers are simultaneously dissociated and denatured.

  Pathogenic conformers can be dissociated and denatured simultaneously using high concentrations of salts or chaotropic agents, eg, guanidinium salts such as about 3M to about 6M guanidinium thiocyanate (GdnSCN), or guanidinium HCl (GdnHCl). Can be done. The chaotropic agent is preferably removed or diluted before the detection is performed because the chaotropic agent can interfere with the binding of the detection reagent.

  In other embodiments, by changing the pH, for example by increasing the pH to 12 or higher (“high pH”) or decreasing the pH to 2 or lower (“low pH”). The pathogenic conformer is simultaneously dissociated and denatured from the complex with the capture reagent. Exposure of the complex to high pH is preferred. In general, a pH of 12.0 to 13.0 is sufficient; preferably a pH of 12.5 to 13.0; more preferably a pH of 12.7 to 12.9; most preferably 12. A pH of 9 is used. Alternatively, exposure of the complex to low pH can be used to dissociate and denature the pathogenic prion protein from the reagent. A pH of 1.0 to 2.0 is sufficient for this alternative. In some embodiments, the pathogenic conformer is treated at a pH of 12.5 to 13.2 for a suitable time, for example, at 90 ° C. (C) for 10 minutes.

  Exposure of the first complex to either high or low pH is generally carried out for a very short time, for example 60 minutes, preferably 15 minutes or less, more preferably 10 minutes or less. In some embodiments, the exposure is performed above room temperature, for example at about 60 ° C., 70 ° C., 80 ° C., or 90 ° C. After exposure for sufficient time to dissociate the pathogenic conformer, either acidic reagent (if high pH dissociation conditions are used) or basic reagent (if low pH dissociation conditions are used) By addition, the pH can be easily readjusted to neutral (ie a pH of about 7.0-7.5). One of ordinary skill in the art can readily determine an appropriate protocol and examples are described herein.

  In general, the addition of NaOH to a concentration of about 0.05 N to about 0.2 N is sufficient to produce high pH dissociation conditions. Preferably, NaOH is added at a concentration of about 0.05N to about 0.15N; more preferably about 0.1N NaOH is used. Once dissociation is complete, the pH can be readjusted to neutral (ie, about 7.0-7.5) by the addition of a suitable amount of an acidic solution such as phosphoric acid, monosodium phosphate.

In general, the addition of H ₃ PO ₄ to a concentration of about 0.2M to about 0.7M is sufficient to produce low pH dissociation conditions. _Preferably, the addition of H 3 PO ₄ at a concentration of 0.3M～0.6M; more preferably, 0.5M H ₃ PO ₄ is used. Once the dissociation is complete, the pH can be readjusted to neutral (ie, about 7.0-7.5) by the addition of a suitable amount of a basic solution such as NaOH or KOH.

  If desired, dissociation of the pathogenic conformer from the complex can be achieved without denaturing the protein, eg, at low concentrations (eg, 0.4-1.0 M) of guanidinium hydrochloride or guanidinium isothiocyanate. Can also be implemented. See WO2006076497 (International Application PCT / US2006 / 001090) for further conditions for dissociating pathogenic conformers from the complex without denaturing the protein. Instead, if the reagent is modified to contain an activatable reactive group (eg, a photoreactive group) that can be used to covalently link the reagent to the pathogenic conformer, The conformer can also be denatured without dissociation from the reagent.

  After dissociation, the pathogenic conformer is then separated from the capture reagent. This separation involves the retention of the unbound sample material described above, except that the portion containing unbound material (currently dissociated pathogenic conformers) is retained and the portion containing the capture reagent is discarded. It can be carried out in the same manner as the removal.

(C. Detection of captured pathogenic conformers)
Detection of pathogenic conformers can be performed using conformational disease protein specific binding reagents. In a preferred embodiment, the CDPSB reagent is an antibody (monoclonal or polyclonal) that recognizes an epitope on a conformational disease protein.

  Detection of captured pathogenic conformers in the sample can also be performed by using a PCSB reagent. Such reagents can be used in embodiments where the capture reagent is either the same or different pathogenic conformer specific binding reagent or conformational disease protein specific binding agent.

  When the method utilizes a first pathogenic conformer specific binding reagent and a second pathogenic conformer specific binding reagent, the first and second reagents may be the same or different. “Same” means that the first and second reagents differ only by the inclusion of a detectable label in the second reagent. The first and second reagents are “different” if, for example, they have different structures or are derived from fragments from different regions of the prion protein.

(General detection method)
Any suitable detection means can then be used to identify the binding between the capture reagent and the pathogenic conformer.

  Analytical methods suitable for use in binding detection include: UV / visible spectroscopy, FTIR, nuclear magnetic resonance spectroscopy, Raman spectroscopy, mass spectrometry, HPLC, capillary electrophoresis, surface plasmon resonance spectroscopy, microscopic Includes methods such as electromechanical systems (MEMS), or any other method known in the art.

  Binding can also be detected by the use of labeling reagents or antibodies, often in the form of an ELISA. Suitable detectable labels for use in the present invention include, but are not limited to, radioisotopes, fluorescent agents, chemiluminescent agents, chromophores, fluorescent semiconductor nanocrystals, enzymes, enzyme substrates, enzyme cofactors , Any detectable molecule, including enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (eg, biotin, streptavidin or haptens), and the like. Additional labels include, but are not limited to, labels that use fluorescence, including substances or portions thereof that can exhibit fluorescence in a detectable range. Specific examples of labels that can be used in the present invention include, but are not limited to, horseradish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethylacridinium ester (DMAE) , Texas Red, Luminol, NADPH and β-galactosidase. Further detectable labels can be detected by nucleic acid detection methods including, for example, polymerase chain reaction (PCR), transcription-mediated amplification (TMA), branched DNA (b-DNA), nucleic acid sequence-based amplification (NASBA), and the like. An oligonucleotide tag can be included. Preferred detectable labels include enzymes, particularly alkaline phosphatase (AP), horseradish peroxidase (HRP), and fluorescent compounds. As is well known in the art, enzymes are utilized in combination with a detectable substrate, such as a chromogenic substrate or a fluorogenic substrate, to produce a detectable signal.

  In addition to the use of labeled detection reagents (described above), immunoprecipitation can be used to separate reagents bound to pathogenic conformers. Preferably, immunoprecipitation is facilitated by the addition of a sedimentation accelerator. Precipitation enhancers include moieties that can promote or increase the precipitation of reagents bound to proteins. Such sedimentation accelerators include polyethylene glycol (PEG), protein G, protein A, and the like. When protein G or protein A is used as a sedimentation accelerator, the protein can optionally be bound to beads, preferably magnetic beads. Sedimentation can be further facilitated by the use of centrifugation or the use of magnetic force. The use of such settling accelerators is known in the art.

  For example, Western blots are tags that detect denatured proteins from SDS-PAGE gels in samples obtained from “pull-down” assays (as described herein), typically electroblotted to nitrocellulose or PVDF. A primary antibody is used. The primary antibody is then detected (and / or amplified) by a probe against the tag (eg, streptavidin-conjugated alkaline phosphatase, horseradish peroxidase, ECL reagent, and / or an amplifiable oligonucleotide). Binding is such as peptides having an affinity tag (eg, biotin) that is labeled and amplified by a probe to the affinity tag (eg, streptavidin-conjugated alkaline phosphatase, horseradish peroxidase, ECL reagent, or amplifiable oligonucleotide). Evaluation can also be performed using a detection reagent.

  For example, when pathogenic conformers are detected directly on individual cells (eg, using fluorescent labeling reagents that allow fluorescence-based cell sorting, counting, or detection of specifically labeled cells) Cell based assays can also be used.

  Assays that amplify the signal from the detection reagent are also known. Examples are assays that utilize biotin and avidin, as well as enzyme labeling and mediated immunoassays, such as ELISA assays. Further examples include the use of branched DNA for signal amplification (eg, US Pat. Nos. 5,681,697; 5,424,413; 5,451,503; 5,4547, No. 025; and 6,235,483); use of target amplification techniques such as PCR, rolling circle amplification, Third Wave invader (Arruda et al., 2002 Expert. Rev. Mol. Diagnostic. 2: 487; US Pat. Nos. 6,090,606, 5,843,669, 5,985,557, 6,090,543, 5846717), NASBA, TMA, etc. (US Pat. No. 6,511,809; EP 0544212A1); and / or immunization PCR techniques (eg US Pat. No. 5,665,539; International Publication W 98/23962; WO00 / 75663; and including the reference) WO01 / 31056.

  In addition, microtiter plate procedures similar to sandwich ELISA can be used, for example, pathogenic conformer specific binding reagents or conformational disease protein specific binding reagents as described herein, wherein the protein is a solid support. Used for immobilization (eg, but not limited to, other pathogenic conformer specific binding reagents or conformational diseases). Protein-specific binding reagents (which may have affinity and / or detection labels such as conjugated alkaline phosphatase, horseradish peroxidase, ECL reagents, or amplifiable oligonucleotides) include pathogenic conformers Used to detect.

(Preferred method for detecting dissociated captured pathogenic conformers)
If the pathogenic conformer bound to the capture reagent is dissociated prior to detection, the dissociated pathogenic conformer will be detected in a direct ELISA or antibody sandwich ELISA type assay, described more fully below. Either can be detected with an ELISA type assay. Although the term “ELISA” is used to describe detection by antibodies, the assay is not limited to assays in which the antibody is “enzyme linked”. The detection antibody can be labeled with any of the detectable labels described herein and well known in the immunoassay art. An ELISA such as that described in Lau et al., PNAS USA 104 (28): 115551-11556 (2007) can be performed to quantify the amount of pathogenic conformers that have dissociated from the capture reagent.

The dissociated pathogenic conformer can be prepared for a standard ELISA by passively coating it on a solid support surface. Such passive coating methods are well known and are typically carried out in 100 mM NaHCO ₃ at pH 8 for several hours at about 37 ° C. or overnight at 4 ° C. Other coating buffers (eg, 50 mM carbonate pH 9.6, 10 mM Tris pH 8, or 10 mM PBS pH 7.2) are well known. The solid support can be any of the solid supports described herein or well known in the art, but a preferred solid support is a microtiter plate, such as a 96 well polystyrene plate. When dissociation is performed using a high concentration of chaotropic agent, the concentration of chaotropic agent is reduced by diluting at least about 2-fold prior to coating on the solid support. When dissociation is performed using high or low pH and then neutralized, the dissociated pathogenic conformer can be used for coating without any further dilution. The plate (s) can be washed to remove unbound material.

  If a standard ELISA is performed, then a detectably labeled binding molecule, such as a conformation disease protein specific binding reagent or a pathogenic conformer specific binding reagent (same or different as used for capture) Either reagent) is added. The detectably labeled binding molecule is reacted with any captured pathogenic conformer and the plate is washed and the presence of the labeled molecule is detected using methods well known in the art. As long as the capture reagent is specific for the pathogenic form, the detection molecule need not be specific for the pathogenic form, but may bind to both forms. In a preferred embodiment, the detectably labeled binding molecule is an antibody. Such antibodies are produced by well-known methods that are specific for both pathogenic and non-pathogenic forms of pathogenic conformers or conformational disease proteins, as well as well-known antibodies. Antibody.

  In an alternative embodiment, dissociated pathogenic conformers are detected using an antibody sandwich ELISA. In this embodiment, the dissociated pathogenic conformer is “recaptured” onto a solid support having a first antibody specific for the pathogenic conformer or conformational disease protein. The solid support on which the pathogenic conformer has been recaptured is optionally washed to remove any unbound material, and then a second antibody specific for the conformational disease protein or pathogenic conformer. And a second antibody is contacted under conditions that allow binding to the recaptured pathogenic conformer.

  The first and second antibodies are typically different antibodies and preferably recognize different epitopes of the conformational disease protein. For example, the first antibody recognizes an N-terminal epitope of the conformational disease protein, and the second antibody recognizes an epitope other than the N-terminal, or vice versa. Other combinations of the first and second antibodies can be easily selected. In this embodiment, not the first antibody but the second antibody is detectably labeled.

  When dissociation of the pathogenic conformer from the reagent is performed using a chaotropic agent, the chaotropic agent should be removed or diluted at least 15-fold prior to performing the detection assay. When causing dissociation using high or low pH and neutralization, the dissociated pathogenic conformer can be used without further dilution. When the pathogenic conformer dissociated prior to performing detection is denatured, both the first and second antibodies bind to the denatured conformer.

(Preferred method for detecting non-dissociated trapped pathogenic conformers)
In other exemplary assays, pathogenic conformers conjugated with capture reagents are not dissociated prior to detection. For example, a solid support (eg, a well of a microtiter plate) is bound to a pathogenic conformer specific binding reagent. A sample containing and suspected of containing the non-prion pathogenic conformer is then added to the solid support. After an incubation period sufficient for any pathogenic conformer to bind to the reagent, the solid support can be washed to remove unbound moieties and detectably labeled as described above. A second binding molecule such as a conformation disease protein specific binding reagent or a second same or different pathogenic conformer specific binding reagent is added. Alternatively, conformational disease protein specific binding can be bound to a solid support (eg, coating the wells of a microtiter plate) and detection can be performed using pathogenic conformer specific detection reagents.

(Preferable detection method for pathogenic Alzheimer's disease conformers)
In a preferred embodiment of the method of the invention, a sandwich ELISA is used when pathogenic Alzheimer's disease conformers are detected. After capture of the pathogenic Alzheimer's disease conformer from the sample using the PCSB reagent and removal of the unbound sample, the captured pathogenic Alzheimer's disease conformer is typically treated with, for example, incubation with guanidine thiocyanate or high pH. Depending on the dissociation conditions, it is dissociated and denatured. In a preferred embodiment, when the pathogenic Alzheimer's disease conformer is Aβ, Aβ is complexed with the PCSB reagent at about 90 ° C. or about 80 ° C. with about 0.05 N NaOH or about 0.1 N NaOH. Dissociates from the body and denatures. In a particularly preferred embodiment, Aβ is dissociated and denatured with about 0.1 N NaOH at about 80 ° C. for about 30 minutes.

  To recapture pathogenic Alzheimer's disease conformers, the solid support can be coated with an antibody specific for Alzheimer's disease protein. This captured pathogenic Alzheimer's disease conformer can then be detected using another antibody specific for the detectably labeled Alzheimer's disease protein. Particularly preferred antibodies are 11A50-B10 (Covance), an antibody specific for the C-terminus of Aβ40; 12F4 (Covance), an antibody specific for the C-terminus of Aβ42; 4G8 specific for Aβ amino acids 17-24; 20.1 specific for Aβ amino acids 1-10; and 6E10 specific for Aβ amino acids 3-8. In a preferred embodiment, 20.1 is a capture antibody and 12F4 or 11A50-B10 is used as a detection antibody.

(D. Solid support used in the assay)
In certain embodiments, the PCSB reagent or CDPSB reagent is provided on a solid support. The PCSB reagent or CDPSB reagent can be provided on the solid support prior to contact with the sample, or the reagent can be contacted with the sample and bound to any pathogenic conformer therein. It can be adapted for binding to a solid support (eg by using a solid support comprising a biotinylation reagent and avidin or streptavidin).

  For the purposes of the present invention, a solid support can be any material that is an insoluble matrix, rigid or capable of linking or binding molecules of interest (eg, reagents, conformational disease proteins, antibodies, etc. of the present invention). It can have a semi-rigid surface. Exemplary solid carriers include, but are not limited to, substrates such as nitrocellulose, polyvinyl chloride; polypropylene, polystyrene, latex, polycarbonate, nylon, dextran, chitin, sand, silica, pumice, agarose, cellulose , Glass, metal, polyacrylamide, silicon, rubber, polysaccharides, polyvinyl fluoride, diazotized paper, activated beads, magnetically responsive beads, and solid phase synthesis, affinity separation, purification, hybridization reactions, immunoassays and others Any material commonly used for such applications. The carrier can be particulate or in the form of a continuous surface, membrane, mesh, plate, pellet, slide, disk, capillary, hollow fiber, needle, pin, chip, non-hollow fiber, gel (eg, silica gel) And beads (eg, pore glass beads, silica gel, polystyrene beads optionally crosslinked by divinylbenzene, grafted copoly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked by NN'-bis-acryloylethylenediamine) , Iron oxide magnetic beads, and glass particles coated with a hydrophobic polymer).

  PCSB or CDPSB reagents as described herein can be obtained by standard techniques for binding PCSB or CDPSB reagents, eg, by adsorption, coupling, or by use of a binding pair, eg, covalently. Can be easily bound to a solid support.

  Immobilization to the support can be enhanced by first coupling a PCSB reagent or CDPSB reagent to the protein (eg, when the protein has better solid phase binding properties). Suitable coupling proteins include, but are not limited to, serum albumin, including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and well known to those skilled in the art Including macromolecules such as other proteins. Other reagents that can be used to attach the molecule to the carrier include polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods for coupling these molecules to proteins are well known to those skilled in the art. For example, Brinkley, M .; A. (1992) Bioconjugate Chem. 3: 2-13; Hashida et al. (1984) J. MoI. Appl. Biochem. 6: 56-63; and Anjaneyulu and Staros (1987) International J. Biol. of Peptide and Protein Res. 30: 117-124.

  If desired, the PCSB reagent or CDPSB reagent added to the solid support can be easily functionalized to produce styrene or acrylate moieties, and thus polystyrene, polyacrylate or other polymers such as polyimide, Polyacrylamide, polyethylene, polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene, polyether, epoxy, silica glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose, etc. Allows the inclusion of molecules in In a preferred embodiment, the solid support is a magnetic bead, more preferably a polystyrene / iron oxide bead.

  PCSB reagent or CDPSB reagent can be bound to a solid support by the interaction of a binding pair of molecules. Such binding pairs are well known and examples are described elsewhere herein. One member of the binding pair is coupled to the solid support by the techniques described above, and the other member of the binding pair is bound to the reagent (before, during, or after synthesis). The PCSB reagent or CDPSB reagent thus modified can be contacted with the sample and the interaction with the pathogenic conformer, if present, can occur in solution; Thereafter, the solid support can be contacted with a reagent (or reagent-protein complex). Preferred binding pairs of this embodiment include biotin and avidin, and biotin and streptavidin. In addition to biotin-avidin and biotin-streptavidin, other suitable binding pairs of this embodiment include, for example, antigen-antibody, hapten-antibody, mimetope-antibody, receptor-hormone, receptor-ligand, agonist-antagonist Lectin-carbohydrate, protein A-antibody Fc. Such binding pairs are well known (see, eg, US Pat. Nos. 6,551,843 and 6,586,193), and one skilled in the art can select a suitable binding pair and place them in the book. Has the ability to adapt for use in the invention. When the capture reagent is suitable for binding to a carrier as described above, the sample can be contacted with the capture reagent before or after the capture reagent is bound to the support.

Alternatively, the PCSB reagent or CDPSB reagent can be covalently bound to the solid support using binding chemistry well known in the art. For example, thiols containing PCSB or CDPSB reagents can be directly coupled to solid supports, such as carboxylated magnetic beads, using standard methods known in the art (eg, Chrisey, LA, Lee, G. U. and O'Ferrall, CE (1996) Covalent attachment of synthetic DNA to self-assembled monolayer films, Nucleic Acids Research 24 (15), 3031-30ait. Aikawa, T., Yoshida, T. and Nishimura, H. (1980) .Preparation and characterization. f hetero-bifunctional cross-linking reagents for protein modifications.Chem.Pharm.Bull.29 (4), see 1130-1135). Carboxylated magnetic beads are first coupled to heterobifunctional crosslinkers containing maleimide functionality (BMPH by Pierce Biotechnology Inc.) using carbodiimide chemistry. The thiolated PCSB or CDPSB reagent is then covalently coupled to the maleimide functionality of the BMPH coated beads. When used in embodiments of the detection method of the present invention, the solid support assists in the separation of complexes comprising reagents and pathogenic conformers from unbound samples. A particularly convenient magnetic bead for thiol coupling is Dynabeads ^™ M-270 carboxylic acid by Dynal. The PCSB or CDPSB reagent may also include a linker, such as one or more aminohexanoic acid moieties.

(E. Preferred detection method)
Preferred embodiments are described below.

  In a preferred embodiment, the methods of the present invention use non-prions using PCSB reagents derived from prion protein fragments, including peptoid reagents as described herein that interact preferentially with pathogenic prion proteins. Pathogenic conformers are captured and detected, and the method includes PSCB reagent in a sample suspected of containing non-prion pathogenic conformers, and non-prion pathogenic conformers present. Contacting to form a complex under conditions that allow binding of the PCSB reagent to the non-prion pathogenic conformer; and if there is a non-prion pathogenic conformer in the sample, the PCSB reagent Detecting a non-prion pathogenic conformer by its binding to. Binding of non-prion pathogenic conformers can be detected, for example, by dissociating the complex and detecting non-prion pathogenic conformers with a CDPSB reagent.

  In one embodiment, the captured non-prion pathogenic conformer is a pathogenic conformer associated with Alzheimer's disease, such as Aβ40, Aβ42, or tau. In such cases, the sample is preferably plasma or cerebrospinal fluid. The PCSB reagent is preferably PrP19-30 (SEQ ID NO: 242), PrP23-30 (SEQ ID NO: 243), PrP100-111 (SEQ ID NO: 244), PrP101-110 (SEQ ID NO: 245), PrP154-165 (SEQ ID NO: 246). From PrP226-237 (SEQ ID NO: 247), SEQ ID NO: 14, SEQ ID NO: 50, SEQ ID NO: 68,

などのペプトイド試薬を含む。

Including peptoid reagents.

  In other preferred embodiments, the methods of the invention use PCSB reagents derived from prion protein fragments, including peptoid reagents as described herein, that interact preferentially with pathogenic prion proteins. Non-prion conformers are captured and non-prion conformers are detected using CDPSB reagents. The method involves contacting a PCSB reagent with a sample suspected of containing a non-prion pathogenic conformer under conditions that permit binding of the reagent to the non-prion pathogenic conformer, if present. Forming a first complex; contacting the first complex with a CDPSB reagent under conditions that allow binding; and if there is a non-prion pathogenic conformer in the sample Detecting the presence of a non-prion pathogenic conformer by its binding to a CDPSB binding reagent. Typically, unbound sample is removed after forming the first complex and before contacting the first complex with the CDPSB reagent. The CDPSB binding reagent can be a labeled anti-conformational disease protein antibody.

  In another yet another preferred embodiment, the method of the invention is derived from a prion protein fragment comprising a peptoid as described herein that interacts preferentially with a pathogenic prion protein and a CDPSB reagent. PCSB reagent is used to capture non-prion pathogenic conformers and detect their presence. The method involves contacting a sample suspected of containing a non-prion pathogenic conformer with a PCSB reagent, if present, under conditions that allow binding of the PCSB reagent to the non-prion pathogenic conformer. Forming a first complex; removing unbound sample material; dissociating the non-prion pathogenic conformer from the first complex and thereby dissociated non-prion Providing a pathogenic conformer; contacting the dissociated non-prion pathogenic conformer with a CDPSB reagent under conditions that permit binding to form a second complex; Detecting the presence of a non-prion pathogenic conformer by detecting the formation of a second complex if a non-prion pathogenic conformer is present in the sample. The formation of the second complex is preferably detected using a detectably labeled second CDPSB reagent, and the PCSB reagent is preferably coupled to magnetic beads.

  In an alternative, the present invention uses a first PCSB reagent derived from a prion protein fragment that includes a peptoid reagent described herein that interacts preferentially with a pathogenic prion protein. Provided is a method for capturing a conformer and detecting a non-prion pathogenic conformer using a second PCSB reagent as described herein. The method allows binding of a sample suspected of containing a non-prion pathogenic conformer with a first PCSB reagent and, if present, the first reagent to the non-prion pathogenic conformer. Contacting under conditions to form a first complex; a sample suspected of containing a non-prion pathogenic conformer, a second PCSB reagent having a detectable label; Contacting under conditions that allow binding of the second reagent to the non-prion pathogenic conformer in the first complex; and if there is a non-prion pathogenic conformer in the sample; Detecting a non-prion pathogenic conformer by its binding to a second reagent.

  In yet another alternative, the present invention provides a peptoid as described herein that uses CDPSB reagents to capture non-prion pathogenic conformers and preferentially interact with pathogenic prion proteins. Methods are provided for detecting non-prion pathogenic conformers using PCSB reagents derived from prion protein fragments, including reagents. The method comprises (a) a sample suspected of containing a non-prion pathogenic conformer under conditions that allow binding of the reagent to the CDPSB reagent and, if present, to the non-prion pathogenic conformer. Contacting to form a complex; (b) removing unbound sample material; (c) a PCSB reagent comprising a label capable of detecting the complex, and a non-prion pathogenic conformer Contacting under conditions that allow binding of the PCSB reagent to the sample; and if there is a non-prion pathogenic conformer in the sample, the binding to the PCSB reagent causes the non-prion pathogenic conformer to The PCSB reagent is preferentially interacting with the pathogenic prion protein from the prion protein fragment.

  In all the above methods, “unbound sample” refers to the components in the sample that have not been captured in the contacting step. Unbound samples can be removed by methods well known in the art, such as by washing, centrifugation, filtration, magnetic separation, and combinations of these techniques. Preferably, in the method of the invention, unbound sample is removed by washing the complex with buffer and / or magnetic separation.

  In a preferred embodiment, the methods of the invention are used for the detection of amyloid diseases including systemic amyloidosis, tauopathy, and synucleinopathies.

(F. Method for detecting Alzheimer's disease)
Methods of detecting pathogenic Alzheimer's disease conformers such as Aβ40, Aβ42, or Tau are provided.

  In particularly preferred embodiments, these methods are derived from prion protein fragments comprising a peptoid reagent described herein that preferentially interacts with pathogenic Alzheimer's disease conformers with pathogenic prion proteins. Captured by the PCSB reagent and the captured conformer is detected by the CDPSB reagent.

In particular, the method includes treating a sample suspected of containing a pathogenic Alzheimer's disease conformer with a PCSB reagent and, if present, conditions that allow the PCSB reagent to bind to the pathogenic Alzheimer's disease conformer. Contacting to form a first complex; removing unbound sample material; dissociating the pathogenic Alzheimer's disease isomer from the first complex and thereby dissociating Providing a pathogenic Alzheimer's disease conformer; contacting the dissociated pathogenic Alzheimer's disease conformer with a CDPSB reagent under conditions that permit binding to form a second complex. And if there is a pathogenic Alzheimer's disease conformer in the sample, the presence of the pathogenic Alzheimer's disease conformer is detected by detecting the formation of a second complex. Comprising the steps of: including a. The pathogenic Alzheimer's disease conformer in the first complex is preferably dissociated and contacted with about 0.05 N NaOH or about 0.1 N NaOH at about 90 ° C. or about 80 ° C. before contacting the CDPSB reagent. Denatured. When the pathogenic Alzheimer's disease conformer is Aβ40 or Aβ42, the pathogenic Alzheimer's disease conformer is preferably dissociated and denatured with about 0.1 N NaOH at about 80 ° C. for about 30 minutes.
Dissociation and / or denaturation can be performed using the methods described in Section IV (B). Typically, pathogenic Alzheimer's disease conformers are simultaneously dissociated and denatured by changing the pH from a low pH to a high pH or from a high pH to a low pH.

  In a preferred embodiment, the PCSB reagent is coupled to a solid support such as magnetic beads, PrP19-30 (SEQ ID NO: 242), PrP23-30 (SEQ ID NO: 243), PrP100-111 (SEQ ID NO: 244), PrP101- 110 (SEQ ID NO: 245), PrP154-165 (SEQ ID NO: 246), PrP226-237 (SEQ ID NO: 247), SEQ ID NO: 14, SEQ ID NO: 50, SEQ ID NO: 68, or

に由来する。

Derived from.

  The CDPSB reagent is preferably an anti-Alzheimer's disease protein antibody coupled to a solid support such as a microtiter plate and the formation of the second complex preferably uses a second detectably labeled CDPSB reagent Is detected. When the pathogenic Alzheimer's disease conformer is Aβ40 or Aβ42, a preferred anti-Alzheimer's disease protein antibody is 11A50-B10 (Covance), an antibody specific for the C-terminus of Aβ40; specific for the C-terminus of Aβ42 It includes the antibody 12F4 (Covance); 4G8 specific for Aβ amino acids 18-22; 20.1 specific for Aβ amino acids 1-10; and 6E10 specific for Aβ amino acids 3-8. In a particularly preferred embodiment, 20.1 is a capture antibody on an ELISA plate and 12F4 or 11A50-B10 is used as the second detectably labeled CDPSB reagent. The sample is preferably plasma or cerebrospinal fluid (CSF).

  Thus, in a particularly preferred embodiment, the method for detecting the presence of pathogenic Alzheimer's disease conformers is not limited to: suspected of containing pathogenic Alzheimer's disease conformers A sample of plasma or CSF is contacted with peptoid XIIb coupled to magnetic beads under conditions that allow binding of peptoid XIIb to a pathogenic Alzheimer's disease conformer, if present. Forming a complex; removing unbound sample material; dissociating the pathogenic Alzheimer's disease isomer from the first complex by changing the pH to dissociate the pathogenic Alzheimer's disease dissociated. Providing a conformer; and an anti-Alzheimer binding the dissociated pathogenic Alzheimer's disease conformer to a solid support Contacting with a protein antibody under conditions that allow binding to form a second complex; and by incubating with a second labeled anti-Alzheimer's disease protein antibody Detecting formation;

(G. Competition assay)
In some embodiments, the methods of the invention detect pathogenic conformers by competitive binding. The detection means can be used to determine when a ligand that binds weakly to the PCSB binding reagent is displaced by a pathogenic conformer. For example, the PCSB reagent can be adsorbed on a solid support. Subsequently, the solid support is bound to a detectably labeled ligand that binds to the PCSB reagent with a weaker binding affinity than the pathogenic conformer binds to the PCSB reagent. A ligand-PCSB reagent complex is detected. The sample is then added. Since the binding affinity of the detectably labeled ligand is weaker than the binding affinity of the pathogenic conformer to the PCSB reagent, the pathogenic conformer displaces the labeled ligand and the label attached to the PCSB reagent A decrease in the detected amount of ligand indicates that a complex has formed between the PCSB reagent and the pathogenic conformer in the sample.

  Thus, in certain embodiments, the presence of a non-prion pathogenic conformer comprises providing a solid support comprising a PCSB reagent; and binding the solid support to a detectably labeled ligand comprising: The binding affinity of the PCSB reagent to the detectably labeled ligand is lower than the binding affinity of the PCSB reagent to the non-prion pathogenic conformer; and containing the non-prion pathogenic conformer A sample suspected of binding to a solid support under conditions that displace the ligand by binding the non-prion pathogenic conformer to the PCSB reagent when a non-prion pathogenic conformer is present in the sample Detecting the complex formed between the PCSB reagent and the non-prion pathogenic conformer from the sample; and detecting P SB reagents are derived from prion protein fragments, preferentially interact with pathogenic prion protein.

(V. Other methods)
In general, the PCSB reagents described herein are capable of preferentially interacting with pathogenic conformers of conformational disease proteins. Therefore, these reagents cause pathogenesis in virtually any biological or non-biological sample, including blood as well as living or dead brain, spinal cord, and other nervous tissue. Allows immediate detection of the presence of sex conformers. Reagents are therefore useful for a wide range of isolation, purification, detection, diagnostic and therapeutic applications.

  For example, the reagents described herein can be used to isolate pathogenic conformers using an affinity carrier. The reagent can be bound to the solid support by, for example, adsorption, covalent bonding, etc. so that the reagent retains its pathogenic conformer selective binding activity. For example, a spacer group may optionally be included so that the reagent binding site is still accessible. The immobilized molecules can then be used to bind pathogenic conformers from biological samples such as blood, plasma, brain, spinal cord, and other tissues. The bound reagent or complex is recovered from the carrier, for example by a change in pH, ie the pathogenic conformer can be dissociated from the complex.

  Thus, in certain embodiments, the present invention relates to binding of a sample suspected of containing a non-prion pathogenic conformer with a PCSB reagent and, if present, a non-prion pathogenic conformer. Contacting under conditions that allow for the formation of a complex; and binding of the pathogenic conformer to the reagent to form a non-prion pathogenic conformer and a non-prion non-pathogenic conformer Providing a method for distinguishing between a non-prion pathogenic conformer and a non-prion non-pathogenic conformer; the PCSB reagent is derived from a prion protein fragment and is pathogenic. Preferentially interacts with prion protein.

  In other embodiments, the present invention allows binding of a sample suspected of containing a non-prion pathogenic conformer with a PCSB reagent and, if present, a non-prion pathogenic conformer. Contacting under conditions to form a complex; and if there is a non-prion pathogenic conformer in the sample, detecting the non-prion pathogenic conformer by its binding to a reagent Providing a method of diagnosing a non-prion conformational disease when a non-prion pathogenic conformer is detected; and wherein the PCSB reagent is derived from a prion protein fragment. Interact preferentially with pathogenic prion proteins.

  Multiple variations and combinations using the reagents described herein may be utilized in the methods of the invention. The following non-limiting examples are described for purposes of illustration.

(Example)
(Example 1: PSR1 binds preferentially to PrP ^Sc )
This example shows that PSR1 (peptoid reagent XIIb bound to magnetic beads (streptavidin M-280 Dynabeads)) selectively pulls down PrP ^Sc .

vCJD or normal brain homogenate (BH) was spiked into 50% pooled normal human plasma in TBS (Tris buffered saline) containing 1% Tween20 and 1% Triton-X100. No BH was added to the control sample. 100 μL of each sample (with or without 10 nL of 10% BH) was mixed with 3 μL of XIIb beads (30 mg / mL), and the resulting mixture was incubated at 37 ° C. for 1 hour with constant shaking at 750 rpm. Unbound sample material was removed by washing the beads four times with TBST containing 0.05% Tween20. At each wash, TBST was added, the beads were collected using magnetic force, and the wash buffer was removed. The PrP ^Sc bound to the beads was dissociated by the addition of 0.1N NaOH. The denatured prion protein was then neutralized with 0.3M NaH ₂ PO ₄ and transferred to an ELISA plate.

  The pull-down efficiency was calculated by comparing the signal from the pull-down sample with the signal from the same sample modified with guanidinium thiocyanate (GdnSCN) without any pull-down. Prion proteins from vCJD or normal brain were denatured by mixing with the same amount of 5% BH and 6M GdnSCN and incubated for 10 minutes at room temperature. The sample was then diluted with TBST to the same concentration as the pull-down sample, with TBST alone as the control. 100 μL of each directly denatured sample was then transferred to the same ELISA plate as the pull-down sample.

The ELISA plate was coated with 2.5 ug / mL anti-prion antibody 3F4 in 0.1 M NaHCO ₃ . The coating procedure was performed overnight at 4 ° C. and then washed 3 times with TBST. The plate was then blocked with 1% casein in TBS for 1 hour at 37 ° C. Prion proteins from both pull-down and directly denatured samples were incubated for 1 hour at 37 ° C. with constant shaking at 300 rpm in an ELISA plate containing 3F4, and the plates were washed 6 times with TBST. Alkaline phosphatase (AP) conjugated detection antibody was diluted to 0.1 μg / mL with 0.1% casein in TBST and then added to the ELISA plate. The plate was then incubated for 1 hour at 37 ° C. and washed 6 times with TBST. Signals were generated using an enhanced Lumi-Phos Plus chemiluminescent substrate and read on a luminometer as relative light units (RLU).

  Results are shown below.

脳組織からのプリオンタンパク質は、３Ｍ ＧｄｎＳＣＮによって完全に変性され得、抗プリオン抗体によって検出することができる。この実験では、発明者らは、ＸＩＩｂビーズを使用したプリオンタンパク質プルダウンによって生成されたシグナルをＧｄｎＳＣＮによる直接変性タンパク質から得たシグナルと比較した。データは、プルダウン試料および直接変性試料のバックグラウンド（ＢＨなし）はそれぞれ９．０および７．７ＲＬＵであることを示した。１０％正常ＢＨの直接変性１０ｎＬは、１４．６ＲＬＵのシグナルを有し、これは、正常脳でのＰｒＰ^ｃレベルを反映していた。一方で、プルダウン法によって検出された１０％正常ＢＨの１０ｎＬは、９．９ＲＬＵの読取値を示し、これはそのバックグラウンドと同様であった。このことは、ペプトイドＸＩＩｂの特異性を示した。１０ｎＬの１０％ｖＣＪＤ試料をプルダウン法および直接変性法によって試験したときに、データは５３．０および５６．３ＲＬＵを示し、このことはＸＩＩｂビーズのプルダウン効率がほぼ１００％に達したことを意味する。

Prion protein from brain tissue can be completely denatured by 3M GdnSCN and can be detected by anti-prion antibodies. In this experiment, we compared the signal generated by prion protein pull-down using XIIb beads with the signal obtained from the directly denatured protein by GdnSCN. The data showed that the background of the pull-down sample and the directly denatured sample (no BH) was 9.0 and 7.7 RLU respectively. 10 nL of direct denaturation of 10% normal BH had a signal of 14.6 RLU, reflecting PrP ^c levels in normal brain. On the other hand, 10 nL of 10% normal BH detected by the pull-down method showed a reading of 9.9 RLU, which was similar to the background. This indicated the specificity of peptoid XIIb. When a 10 nL 10% vCJD sample was tested by pull-down and direct denaturation methods, the data showed 53.0 and 56.3 RLU, which means that the pull-down efficiency of XIIb beads has reached almost 100% .

Example 2 PSR1 also interacts preferentially with aggregated amyloid beta protein (Aβ)
This example

ＰＳＲ１はまた、凝集Ａβと優先的に相互作用することを示す。

PSR1 also shows preferential interaction with aggregated Aβ.

  This example demonstrates the presence of insoluble aggregated Aβ40 / 42 in multiple Alzheimer's disease brains, and that detection of these aggregates can only be achieved after the antibody epitope masked in the aggregates has been exposed by denaturation. Indicates. PSR1 capture of aggregated Aβ from these brains is specific and is mediated by peptoid XIIb rather than pull-down beads. This example shows that PSR1 selectively captures aggregated Aβ40 / 42 spiked in plasma, a sample matrix containing soluble Aβ40 / 42 that is not recognized by PSR1. Finally, this example shows that PSR1 captures aggregated Aβ40 / 42 by showing that capture was hindered by denaturation of the sample with a chemical denaturant prior to pull-down.

(Quantification of total Aβ40 / 42 in Alzheimer's disease brain and normal brain)
Total Aβ40 / 42 in the brain was quantified using a sandwich ELISA using antibodies commercially available from Covance. Individual wells of a 96 well capture plate were coated with either mAb 11A50-B10 (detecting only soluble Aβ40) or mAb 12F4 (detecting only soluble Aβ42). Aβ40 or Aβ42 captured on the ELISA plate was detected by mAb4G8 (recognizing all forms of Aβ containing the sequence VFFAE) conjugated to alkaline phosphatase (AP). Lumiphos Plus (Lumigen) was used as a chemiluminescent substrate. Chemiluminescence was read with a Luminoskan luminometer (Thermo).

  Aβ40 and Aβ42 levels of AD (patient identification numbers 325-1, 334, 325, 264, 230, 218, 221, 201, 184, 177, 291) and control (patients 320, 326, 327, 328) brain homogenates are This was determined using this sandwich ELISA detection system. Denaturation of brain homogenate with 5.4M GdnSCN was necessary to detect the majority of Aβ peptides (FIGS. 1, 5B, and 5E). It is well accepted that antibodies will not bind to epitopes that are conformationally modified or masked in aggregates. This property has been observed previously with several antibodies that recognize prions and other amyloid proteins. The ELISA data indicates that pathogenic aggregate Aβ has accumulated in AD brain but not in age-matched non-AD control brain. The control brain did not contain any high levels of either Aβ form (soluble or aggregated), but both Aβ40 and Aβ42 were readily detected from AD patient samples (FIG. 1).

  Dose setting of one AD brain homogenate (patient number 291) detected by ELISA in parallel with a standard curve (denatured synthetic Aβ peptide) allows determination of the concentration of Aβ40 and Aβ42 in this brain homogenate (FIG. 5A). , B and 5D, E), calculated as 10 and 240 μg / mL, respectively.

(Aβ40 / 42 in AD brain is aggregated)
Since detection by sandwich ELISA was improved by treatment with denaturing agent prior to ELISA, Aβ from AD BH was considered aggregated (FIG. 1). To test whether this Aβ is insoluble (another feature of aggregates), AD BH was centrifuged and the supernatant and pellet fractions were utilized for ELISA (FIG. 2). In native AD BH, most of the Aβ signal was in the pellet fraction. However, pretreatment of BH with GdnSCN shifted the Aβ signal to the supernatant fraction. This experiment shows that Aβ aggregates found in AD BH are insoluble under our centrifugation conditions and that Aβ is soluble by pretreatment with chemical denaturants. Thus, by directly examining the physical properties of Aβ in AD BH, the inventors have confirmed that most of the Aβ peptide from AD BH is aggregated. This is a well-known phenomenon that has been observed in a class of diseases, described as amyloid diseases. A few examples of these diseases include prion disease (eg CJD), AD, Parkinson's disease, type II diabetes, and systemic amyloidosis. It is related to the conversion from normal conformation to β-sheet conformation.

(PSR1 binding to Aβ is mediated by peptoid reagent XIIb)
PSR1 captured Aβ from AD brain homogenate spiked into 80% plasma in TBSTT buffer (FIG. 3). Brain homogenates from AD and control brains were incubated with PSR1 beads or control glutathione beads (no peptoid XIIb). The beads were washed and the capture material was dissociated and denatured from the beads with 6M GdnSCN. The beads were removed using a magnet to dilute the denatured sample and added to an ELISA plate pre-coated with anti-Aβ42 antibody 12F4. Detection was performed using AP-labeled 4G8 antibody. While PSR1 captured Aβ from AD samples, control beads (M270-carboxylic acid beads reacted with maleimide and glutathione (M270-glutathione produced in-house)) produced detectable amounts of Aβ from any brain sample. Could not be captured. This experiment confirms that the ability to capture Aβ is not due to the beads themselves, but to the peptoid XIIb covalently bound to the beads.

(PSR1 selectively binds to aggregated Aβ in the presence of soluble Aβ)
Plasma contains significant levels of soluble Aβ40 and Aβ42. Therefore, to assess whether PSR1 can selectively bind to aggregated Aβ in the presence of soluble Aβ, brain homogenate from AD patient number 291 was spiked into plasma and subjected to PSR1 pull-down.

  4A and 4B show the quantification of endogenous Aβ levels using our sandwich ELISA in increasing concentrations of plasma diluted in TBST (normal human plasma from a commercial source). Soluble Aβ40 and Aβ42 levels were found to be in the range of 10-100 ng / ml.

  Initially, an ELISA was performed on native and denatured AD BH patient number 291 samples to assess the amount of aggregated Aβ40 / 42 present in the samples (FIG. 5B-Aβ42; FIG. 5D-Aβ40).

  Samples with increasing amounts of AD brain homogenate spiked into plasma (80% plasma in TBSTT buffer) were then subjected to PSR1 pulldown. PSR1 was able to selectively capture Aβ42 and Aβ40 from spiked AD brain homogenates (10, 20, 50 nL spikes), but not any soluble Aβ42 or Aβ40 from plasma alone (0 nL spikes). None (see FIGS. 5C and F, open triangle: PSR1-natural Aβ). This suggests that PSR1 captures only aggregated Aβ peptide found in Alzheimer's disease brain and not soluble Aβ found in plasma. This result also shows that plasma components, which contain high concentrations of various proteins, lipids, and ions, do not interfere with PSR1 binding.

(PSR1 capture is hindered by solubilization of the sample before pull-down)
When the same AD brain homogenate was denatured with 5.4 GdnSCN to solubilize aggregates prior to incubation with plasma and PSR1, no Aβ was detected. (See FIG. 5C and F-gray triangle: PSR1-modified Aβ). This means that PSR1 recognizes the misfolded aggregation properties of Aβ found in AD samples that can be solubilized by denaturation, but does not recognize another determinant specific for AD-derived Aβ peptides. Support the idea.

(Example 3 Peptides derived from prion protein fragments have similar capture profiles for pathogenic prion protein and aggregated Aβ from diseased brain homogenates spiked in buffer or plasma)
WO05 / 016137, WO07 / 030804 describe various peptides and peptoids derived from prion protein fragments that interact preferentially with pathogenic conformers of prion protein. This experiment suggests that these reagents capture Aβ by a similar mechanism that these reagents capture pathogenic prions.

  Six different peptides from prion fragments with three different capture profiles were tested. The amino acid sequence of each peptide corresponds to a fragment of the human prion protein sequence and is described below. The subscript indicates the amino acid position of the first and last amino acid of the fragment.

１）グループ１：ＰｒＰ_{１９−３０}およびＰｒＰ_{１００−１１１}、これらは、血漿中および緩衝液中の両方でＰｒＰ^Ｓｃを捕捉する。
２）グループ２：ＰｒＰ_{１５４−１６５}およびＰｒＰ_{２２６−２３７}、これらは、緩衝液中でのみＰｒＰ^Ｓｃを捕捉できる。
３）グループ３：ＰｒＰ_{３７−４８}およびＰｒＰ_{１８１−１９２}、これらは、ＰｒＰ^Ｓｃを捕捉できないペプチドである。ＰｒＰ_{３７−４８}およびＰｒＰ_{１８１−１９２}はＰｒＰ_{１５４−１６５}およびＰｒＰ_{２２６−２３７}と同様の物理化学特性を有するが、異なるアミノ酸配列を有するペプチドであるため、これらを負の対照として選択した。

1) Group 1: PrP _19-30 and PrP _100-111 , which capture PrP ^Sc both in plasma and in buffer.
2) Group _{2: PrP 154-165} and _{PrP 226-237,} these can capture ^{PrP Sc} only in buffer.
3) Group 3: PrP _37-48 and PrP _181-192 , these are peptides that cannot capture PrP ^Sc . PrP _37-48 and _{PrP 181-192} has a similar physicochemical properties as _{PrP 154-165} and _{PrP 226-237,} for a peptide having a different amino acid sequence, and select these as a negative control.

The result was very surprising. The activity profile of aggregated Aβ42 captured by the peptides was almost the same as the profile of captured PrP ^Sc from patients suffering from the prion-based disease Creutzfeldt-Jakob disease (FIG. 6): Aggregated Aβ42 spiked into either plasma or buffer was captured; Group 2 peptides captured aggregated Aβ42 spiked into buffer, but did not capture aggregated Aβ42 spiked into plasma; Group 3 peptides Did not capture either Aβ42. This result is very strong evidence that the binding mechanism between the tested PCSB reagent and PrP ^Sc binding and the binding of PCSB reagent and Aβ aggregates is similar. The most promising interaction domain is a motif that is common and part of the general amyloid structure, regardless of protein amino acid sequence.

(Overview)
Both this example and Example 2 show that reagents that can interact preferentially with pathogenic prions can also interact preferentially with aggregated Aβ. The ability of these reagents to bind both pathogenic prions and aggregated Aβ with such similar binding characteristics was completely unexpected.

  The ability of these reagents to capture aggregated proteins allows direct detection of Alzheimer's disease associated Aβ protein aggregates. This is advantageous compared to testing for more indirect markers of AD disease. This is also advantageous compared to reagents such as anti-Aβ antibodies that specifically bind to non-aggregated Aβ. Such antibodies can only associate with soluble Aβ, present in normal and AD patients, whose concentration in a biological fluid is the only indirect marker of AD disease. Using such a reagent to detect aggregated Aβ requires comparison of the natural sample with a sample that has been treated to solubilize the aggregate. Unfortunately, this analysis does not work with samples containing very low levels of aggregates and requires sample manipulation that tends to dilute Aβ levels beyond the detectable limit.

Example 4 Effect of Sample Matrix on Aggregated Aβ42 Pulldown
The effect of the sample matrix on the pull-down of aggregated Aβ42 showed that PSR1 and the peptides described above were spiked into 3 different Alzheimer's disease brain samples: 1) brain homogenate spiked in buffer; 2) brain homogenate spiked in plasma; Evaluated by testing with brain homogenates spiked into CSF (see Figure 7).

  Samples were prepared and processed as described in FIG. Brain homogenates spiked into CSF were prepared by spiking 50 nL of BH into 100 microliters of 50% plasma in TBSTT buffer.

  This experiment confirms other studies showing that PSR1 interacts preferentially with aggregated Aβ42 in the presence of plasma and shows that PSR1 also interacts preferentially with aggregated Aβ42 in the presence of CSF. ing.

The peptides tested showed various capture profiles. Differences in capture behavior in buffer, plasma, and CSF suggest that there are interfering components in plasma and CSF that interfere with the binding mechanism. A wider range of reagents can capture aggregated Aβ42 in CSF compared to plasma. PrP _19-30 and PrP _100-111 , which can capture Aβ42 in the presence of plasma, can also capture Aβ42 in the presence of CSF. However, _{PrP 154-165} and _{PrP 226-237} unable capture Aβ in the presence of plasma can capture Aβ in the presence of CSF. This finding is consistent with the fact that CSF is a less complex sample matrix with a lower protein concentration than plasma. Therefore, it is less likely to contain components that can interfere with the interaction between the reagent and aggregated Aβ.

In summary, these experiments show that the positively charged reagents _PSR1 , PrP _19-30 , and PrP _100-111 are fluids that can be applied by pre- _mortem diagnosis of Alzheimer's disease as well as simple buffer (TBSTT), For example, it was shown that CSF and plasma could interact with aggregated Aβ.

Example 5 Dissociation and denaturation of Aβ for ELISA-Optimization of NaOH concentration and incubation temperature
Brain homogenates (BH) from normal or Alzheimer's disease (AD) patients were spiked into normal human plasma (NHP) and captured by PSR1 beads. After washing, the beads were resuspended with NaOH (0.01N-0.3N) in a PCR thin-walled tube and incubated for 10 minutes at 60 ° C, 70 ° C, 80 ° C, or 90 ° C on a Perkin Elmer MasterCycler. The denatured Aβ sample was neutralized to about pH 7.5 and then proceeded to sandwich ELISA detection.

  The results showed the maximum signal when Aβ was denatured with 0.05N NaOH at 90 ° C., but the dynamic range was small (see FIG. 8). When denatured at 80 ° C., the Aβ signal reached a plateau from 0.05 N to 0.15 N NaOH, with 0.1 N NaOH showing the second largest signal. Therefore, further studies focused on denaturing Aβ with 0.1 N NaOH at 80 ° C.

  Aβ requires NaOH denaturation at a higher temperature and longer incubation than the prion protein. For Aβ, denaturation of Aβ with 0.1N NaOH for 30 minutes at 80 ° C. works well, whereas for prions, 0.1N NaOH for 10 minutes at room temperature works well.

Example 6 PSR1 capture / sandwich ELISA for amyloid beta detection
This example describes a detection protocol for Aβ using PSR1 as a pathogenic conformer specific binding (PCSB) reagent. The assay has three basic steps: 1) capture of Aβ aggregates from the sample using the PCSB reagent, 2) denaturation and dissociation of the binding agent from the PCSB reagent using a chaotropic agent, and 3) a commercially available antibody. Having a sandwich ELISA. When 4G8-AP is used as a detection reagent in an ELISA, a linear response superior to 4G8-HRP is obtained.

(Sample preparation)
Spike 100 nL of 10% brain homogenate (BH) into the following 100 μL / assay: a) 50% normal human plasma (NHP) and 1 × TBSTT (50 mM Tris, pH 7.5; 150 mM NaCl; 1% Tween-20; 1) Triton X-100); b) 50% normal human cerebrospinal fluid (CSF) and 1 × TBSTT; or c) 0.1% bovine serum albumin (BSA) and 1 × TBSTT.

(Bead capture)
These samples are added at 30 mg / mL to 3 μL / assay of PSR1 peptoid covalently coupled to Dynal M270-carboxylic acid beads and incubated for 1 hour at 37 ° C. with shaking. Instead, the sample is added to 10 μL / assay of M280-streptavidin beads (10 mg / mL) coated with biotinylated PSR1 peptoid.

  The beads are washed 4 times with 275 μL of TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20). For each wash, the washed beads are collected with a magnet to remove the wash buffer.

  2 μL / assay of 6M guanidine thiocyanate is then added and the sample is incubated for 30 minutes at room temperature to elute and denature the captured material. Then add 98 μL TBST / assay to dilute the guanidine thiocyanate.

  In order to add the correct amount of 6M guanidine thiocyanate, sample preparation and bead capture are done in bulk (multiple reactions in one tube or well). After diluting the GdnSCN, transfer the sample to a 96 well microtiter plate for the remainder of the protocol.

  The beads are removed by magnetic separation and the supernatant is transferred to a capture plate.

(detection)
Capture plate (Microlite 2+ by Thermo Scientific), mouse monoclonal antibody (mAb) 11A50-B10 (C-terminal antibody; specifically binding Aβl-40) or mAb 12F4 (C-terminal antibody; specifically binding Aβ1-42) ) Either at 2.5 μg / mL. Both antibodies are commercially available from Covance.

  The supernatant is incubated on the capture plate for 1 hour at 37 ° C. Wash capture plate 4 times with TBST 275 μL / well.

  0.2 μg / mL mAb4G8 conjugated to alkaline phosphatase in 0.1% BSA and TBST is added to the capture plate for detection. Purified antibodies are available from Covance. AP conjugates are generated in-situ using the starting material (antibody) from Covance.

  The capture plate is then incubated with detection antibody for 1 hour at 37 ° C. and washed 4 times with TBST 275 μL / well.

  Add 100 μL of enhanced Lumiphos Plus (add 0.55% SDS at a ratio of 91 μL per mL of chemiluminescent substrate Lumiphos Plus for detection) to the capture plate. The capture plate is then incubated for 30 minutes at 37 ° C. and the plate is read on a LuminoScan.

Example 7 Synthesis of PCSB reagent used in the method of the present invention
Peptide fragments of prion proteins are essentially as described by Merrifield (1969) Advan. Enzymol. 32: 221 and Holm and Medal (1989), Multiple column peptide synthesis, p. 208E, Bayer and G.C. Jung (ed.), Peptides 1988, Walter de Gruter & Co. Berlin-N. Y. Chemically synthesized using standard peptide synthesis techniques as described in. The peptide was purified by HPLC and the sequence was verified by mass spectrometry.

  In some cases, the synthesized peptides contained additional residues, such as GGG residues, at the N or C terminus and / or contained one or more amino acid substitutions compared to the wild type sequence.

(A. Peptoid substitution)
Peptoid substitutions were performed in SEQ ID NO: 14 (QWNKPSKPKTN, corresponding to residues 97-107 of SEQ ID NO: 2), SEQ ID NO: 67 (KKRPKPGGWNTGG, corresponding to residues 23-36 of SEQ ID NO: 2) and SEQ ID NO: 68 (KKRPKPGGG, SEQ ID NO: 2) (corresponding to residues 23-30 of 2). In particular, one or more proline residues of these peptides were replaced with various N-substituted peptoids. See FIG. 9 or peptoids that can replace any proline. Peptoids are described in US Pat. Nos. 5,877,278 and 6,033,631, both incorporated herein by reference in their entirety; Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89: 9367 prepared and synthesized.

(B. Multimerization)
Certain peptide reagents were also prepared as multimers, for example by preparing tandem repeats (conjugation of multiple copies of a peptide by a linker such as GGG), multi-antigenic peptides (MAPS) and / or linear binding peptides.

  In particular, MAPS is essentially described in Wu et al. (2001) J Am Chem Soc. 2001 123 (28): 6778-84; Spetzler et al. (1995) Int J Pept Protein Res. 45 (1): 78-85 as prepared using standard techniques.

Linear and branched peptides (eg, PEG linker multimerization) were also prepared using standard techniques using a polyethylene glycol (PEG) linker. In particular, a branched multipeptide PEG backbone was generated using the following structures: biotin-PEG-Lys-PEG-Lys-PEG-Lys-PEG-Lys-PEG-Lys (no peptide control) and biotin-PEG- Lys (peptide) -PEG-Lys (peptide) -PEG-Lys (peptide) -PEG-Lys (peptide) -PEG-Lys (peptide). Furthermore, to prepare the binding of Lys of the peptide: Lys- epsilon ^{_{-NH-CO- (CH 2) 3}} -Mal-S-Cys- peptide. See Figure 10C.

(C. Biotinylation)
Peptides were biotinylated using standard techniques after synthesis and purification. Biotin was added to the N or C terminus of the peptide.

Example 8 PCSB Peptoid Reagent
The following PCSB peptoid reagents are synthesized methods for the preparation of peptoid molecules containing N-substituted glycine residues, eg, US Pat. No. 5,811,387, each incorporated herein by reference in its entirety. No. 5,831,005; No. 5,877,278; No. 5,977,301; No. 6,075,121; No. 6,251,433; 6,033,631 as well as Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89: 9367, for example.

(Peptoid Reagent I)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 229.

計算質量：１０５４．４２；実測質量（Ｏｂｓｅｒｖｅｄ Ｍａｓｓ）：１０５４．２。実測質量の測定は全て、Ｗａｔｅｒｓ（Ｍｉｌｆｏｒｄ，ＭＡ）Ｍｉｃｒｏｍａｓｓ ＺＱ ＬＣ／ＭＳシステムで測定した。

Calculated mass: 1054.42; Observed Mass: 1054.2. All measured mass measurements were made with a Waters (Milford, Mass.) Micromass ZQ LC / MS system.

(Peptoid Reagent II)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 230.

計算質量：１２９０．７０；実測質量：１２９０．８。

Calculated mass: 1290.70; measured mass: 1290.8.

(Peptoid Reagent III)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 231.

計算質量：１８６１．３０；実測質量：１８６１．６。

Calculated mass: 1861.30; measured mass: 1861.6.

(Peptoid reagent IV)
Lower peptoid reagent

は、これに限定されるわけではないが配列番号２３２を含む。

Includes, but is not limited to, SEQ ID NO: 232.

(Peptoid Reagent V)
Lower peptoid reagent

は、これに限定されるわけではないが配列番号２３３を含む。

Includes, but is not limited to, SEQ ID NO: 233.

(Peptoid Reagent VI)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 234.

計算質量：１９５６．４９；実測質量：１９５６．２。

Calculated mass: 1956.49; Observed mass: 1956.2.

(Peptoid reagent VII)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 235.

計算質量：１８９６．３９；実測質量：１８９６．４。

Calculated mass: 189.39; observed mass: 1896.4.

(Peptoid reagent VIII)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 236.

計算質量：１７３２．１８；実測質量：１７３２．４。

Calculated mass: 1732.18; observed mass: 1732.4.

(Peptoid Reagent IX)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 237.

計算質量：１２４８．６５；実測質量：１２４８．４。

Calculated mass: 1248.65; measured mass: 1248.4.

(Peptoid Reagent X)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 238.

計算質量：１２４８．６５；実測質量：１２４８．４。

Calculated mass: 1248.65; measured mass: 1248.4.

(Peptoid reagents XIa and XIb)
The lower peptoid reagents XIa and XIb contain SEQ ID NO: 239.

ＸＩａ：計算質量：１３０４．７６；実測質量：１３０４．６。
ＸＩｂ：計算質量：１１６６．５９；実測質量：１１６６．２。

XIa: Calculated mass: 1304.76; observed mass: 1304.6.
XIb: Calculated mass: 1166.59; Observed mass: 1166.2.

(Peptoid reagents XIIa and XIIb)
The lower peptoid reagent Formulas XIIa and XIIb contain SEQ ID NO: 240.

ＸＩＩａ：計算質量：１２７６．７１；実測質量：１２７６．６。

XIIa: calculated mass: 1276.71; observed mass: 1276.6.

(Peptoid Reagent XIII)
The lower peptoid reagent includes, but is not limited to, SEQ ID NO: 241.

計算質量：１２５６．５８；実測質量：１２５６．６。

Calculated mass: 1256.58; observed mass: 1256.6.

(Example 9 PSR1 and PrP23-30 show better Aβ capture than β sheet blockers)
β-sheet breakers are small molecules that are thought to interfere with the Aβ fibrosis process by binding to the region of Aβ that mediates aggregation. This example shows that PSR1 capture of Aβ is superior to capture by a β sheet breaker (see FIG. 11).

  PSR1, PrP23-30, M280SA beads only and β sheet breakers AL30, AL32, AL33, and AL34 (its structure is detailed in the table below)

を使用するＡβの捕捉、および、４Ｇ８−ＨＲＰ試薬を用いたＥＬＩＳＡによる検出は、実施例２に記載した方法を使用して実施した。

Aβ capture using and detection by ELISA using 4G8-HRP reagent were performed using the method described in Example 2.

Example 10 PSR1 captures aggregated total tau)
Total levels of aggregated tau (phosphorylated, non-phosphorylated, or hyperphosphorylated) are associated with Alzheimer's disease. Hyperphosphorylated aggregated tau appears to be particularly strongly associated with Alzheimer's disease.

Tau hyperphosphorylation caused by Aβ aggregation and oxidative stress is thought to be involved in the development of AD (Formichi, P. et al., J. of Cellular Physiology. 208-1: 39-46, 2006). Hyperphosphorylated tau protein has high self-aggregation activity and forms counter-helical fibrils and neurofibrillary tangles found in neurodegenerative disease brains (Goedert, M. et al., Trends Neurosci 16: 460-465. , 1993; Iqbal et al., J Neural Trans 53: 169-180, 1998). Tau phosphorylation at Thr181 and 231 has been tested as an AD biomarker in CSF. The ratio of p- tau against A [beta] ₄₂ is to distinguish patients with AD and healthy controls and other dementias, with the value of the high diagnostic (Buerger et al., Arch Neurol 59: 1267-1272,2002; Maddalena Et al., Neuro Neurol 60: 1202-1206, 2003).

  This example shows that PSR1 captures AD-related aggregated tau.

(Measurement of total tau levels in normal and Alzheimer brains)
Normal human and Alzheimer's disease brain homogenates were treated with 3M GnSCN for 1 hour at room temperature (degeneration) or untreated (native). 100 nL of 10% brain homogenate was dispensed into each ELISA well (BioSource Tau immunoassay kit KHB0042 / KHB0041). All tau standards were diluted with a dilute solution containing 0.006M GnSCN.

  Each ELISA plate was coated and incubated overnight at room temperature. On the second day, the ELISA plate was washed 4 times with 400 uL of diluted wash buffer per well and 100 uL of rabbit anti-tau antibody was added to each well. Plates were incubated for 1 hour at room temperature and washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of functional anti-rabbit HRP was added to each well. The plates were then incubated for 30 minutes at room temperature and washed 4 times with 400 uL of diluted wash buffer per well. Next, 100 uL of Stabilized Chromagen was added. The plates were then incubated for 30 minutes at room temperature in the dark. The reaction was stopped by adding 100 uL of stop solution to all wells and read at 450 nm.

  Significant levels of tau were detected in both normal and Alzheimer's disease brains (Figure 12). Unlike prions and Aβ aggregates, tau denaturation has no significant effect on detectable tau levels, indicating that tau antibody-binding epitopes are conformationally altered in tau pathogenic isoforms. Suggested not.

(Pull down by PSR1)
400 nL of 10% normal or Alzheimer's disease brain homogenate was diluted with 100 uL of TBSTT for each pull-down reaction. 3 uL of M270-glutathione or PSR1 beads per reaction was plated in a 500 uL round bottom coning plate. The reaction was incubated for 1 hour at 37 ° C. with shaking at 750 rpm. The beads were then washed with TBST in BioTek BLx405. After removing the residual solution, 5 uL of 6M GnSCN was added and incubated with the beads at 750 rpm for 1 hour at room temperature to dissociate the protein. 120 uL of H ₂ O was added to each well.

  Next, the amount of tau captured by each pull-down reagent was quantified. 50 uL of standard dilution buffer per well was added to the tau ELISA plate. The pull-down plate was placed on a magnetic stand for 2 minutes and 50 uL / well of the solution was transferred to a tau ELISA plate (equivalent to 160 nL 10% BH / well). The tau standard was diluted with a dilute solution containing 0.12M GnSCN. ELISA plates were coated and incubated overnight at room temperature. On the second day, the ELISA plate was washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of rabbit anti-tau antibody was added to each well. Plates were incubated for 1 hour at room temperature and washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of functional anti-rabbit HRP was added to each well. Plates were incubated for 30 minutes at room temperature and washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of Stabilized Chromagen was added. Plates were incubated for 30 minutes at room temperature in the dark. The reaction was stopped by adding 100 uL of stop solution to all wells and read at 450 nm.

  In most Alzheimer's disease brain samples, PSR1 bound significantly more tau than in normal brain samples, suggesting that PSR1 selectively binds to aggregated tau (FIG. 13). Only a small amount of tau was detected in the pull-down sample of control glutathione beads.

  Table 8 below shows the detected tau level.

で定量する。

Quantify with.

Example 11 Dissociated aggregated tau is no longer pulled down by PSR1 beads (FIG. 14))
Normal or Alzheimer brain homogenates were incubated for 1 hour at room temperature with or without 5M GnSCN. 400 nL of 10% normal or Alzheimer's disease brain homogenate was diluted with 200 uL of 25% human plasma-TBSTT for each pull-down reaction.

3uL of M270-glutathione or PSR1 beads per reaction was placed in a 500uL round bottom Corning plate. The reaction was incubated for 1 hour at 37 ° C. with shaking at 750 rpm. The beads were then washed with TBST in BioTek BLx405. After removing the residual solution, 10 uL of 6M GnSCN was added and incubated with beads at 750 rpm for 1 hour at room temperature to dissociate the protein. 120 uL of H ₂ O was added to each well.

  The pull-down plate was placed on a magnetic stand for 2 minutes and 50 uL / well of the solution was transferred to a tau ELISA plate (equivalent to 160 nL 10% BH / well). ELISA plates were coated and incubated overnight at room temperature. On the second day, the ELISA plate was washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of rabbit anti-tau antibody was added to each well. Plates were incubated for 1 hour at room temperature and washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of functional anti-rabbit HRP was added to each well. Plates were incubated for 30 minutes at room temperature and washed 4 times with 400 uL of diluted wash buffer per well. 100 uL of Stabilized Chromagen was added. Plates were incubated for 30 minutes at room temperature in the dark. The reaction was stopped by adding 100 uL of stop solution to all wells and read at 450 nm.

  The dissociated aggregated tau was no longer bound by PSR1 (see FIG. 14).

現在までに発明者らは、３種類の異なるタンパク質；プリオン、Ａβ、およびタウの不溶性規則的タンパク質凝集体がＰＳＲ１および他のＰＣＳＢ試薬を結合することを示している。これらの凝集体の変性によって、溶解性が生じ、ＰＳＲ１および他のＰＣＳＢ試薬へのその結合を除去する。この観察結果は、タンパク質アミノ酸配列とは無関係の規則的アミロイド構造を特徴とする相互作用ドメインの存在をさらに支持する。

To date, the inventors have shown that insoluble regular protein aggregates of three different proteins; prion, Aβ, and tau bind PSR1 and other PCSB reagents. Denaturation of these aggregates causes solubility and removes its binding to PSR1 and other PCSB reagents. This observation further supports the existence of an interaction domain characterized by a regular amyloid structure independent of the protein amino acid sequence.

Example 12 Limit of Detection (LOD) of PSR1 Bead Pulldown Assay for AD Aβ Aggregates in Human CSF (FIG. 15)
The limit of detection (LOD) of the PSR1 bead pull-down assay was assessed by determining the minimum amount of aggregated Aβ detectable beyond background.

  The sensitivity of the sandwich ELISA to detect monomer soluble Aβ was evaluated using the MesoScale Discovery (MSD) technique (FIG. 15A). Standard synthetic Aβ40 or 42 was captured by mAb specific for the C-terminus of Aβ and detected by mAb 4G8. The detection limit (LOD) with a signal / noise ratio of 2 was 1.6 pg / mL for Aβ40 and 12 pg / mL for Aβ42.

  PSR1 sensitivity to detect Aβ40 and 42 aggregates in Alzheimer's disease (AD) brain homogenate (BH) was measured (FIG. 15B). 10% AD BH was spiked into 200 uL of pooled normal human CSF and captured by 3 uL of PSR1 beads. Captured Aβ was dissociated into monomeric Aβ with 0.1 N NaOH at 80 ° C. and detected by sandwich MSD ELISA as described above. The detection limit (LOD) with a signal / noise ratio of 2 was 1 pg / mL for Aβ40 (10% AD BH 11.4 nL spiked into 1 mL of CSF) and 1 pg / mL for Aβ42 (10% AD BH). 0.3 nL spiked into 1 mL of CSF).

(Example 13 PSR1 recovery of Aβ42 aggregate spiked in human serum (binding efficiency) (FIG. 16))
The recovery efficiency of Aβ aggregates by PSR1 was evaluated by comparing the amount of total aggregated Aβ42 in AD BH with the amount captured by PSR1 pulldown.

  10% AD BH was spiked into 200 uL of diluted normal human serum (200-fold diluted with TBS) and captured by 3 uL of PSR1 beads (FIG. 16, square). Captured Aβ42 aggregates were dissociated with 0.1 N NaOH at 80 ° C. and detected by sandwich MSD ELISA as described in Example 12.

  The same amount of AD BH that was not subjected to PSR1 capture was denatured with 0.1 NaOH at 80 ° C. and total Aβ42 was measured by sandwich ELISA as described in Example 12 (FIG. 16, triangles).

  The concentration of Aβ was calculated from a standard curve using synthetic Aβ as described in Example 12.

  The amount of Aβ42 aggregates captured by PSR1 is equal to the total Aβ42 aggregates in AD BH, indicating that PSR1 recovery is about 100% and that PSR1 is a very efficient capture reagent. It was.

Example 14 Detection of Aβ40 and 42 from normal CSF by PSR1 bead pull-down assay
To monitor the ability of PSR1 to bind Aβ monomer in CSF from individuals not suffering from Alzheimer's disease, increasing concentrations of PSR1 (3, 9, 15 uL from a 30 mg / mL stock) were added 2 × TBSTT (1% Tween CSF lot 410 or lot 411 50 uL mixed with 50 uL of Tris-buffered saline containing 20 and 1% Triton-X 100). The resulting mixture was incubated at 37 ° C. for 1 hour with constant shaking at 600 rpm. Unbound samples were removed by washing the beads 6 times with TBST (Tris buffered saline containing 0.05% Tween 20). For each wash, after addition of TBST, beads were collected using magnetic force to remove TBST. The bead-bound protein was dissociated with 0.1 N NaOH for 30 minutes at 80 ° C. with constant shaking at 750 rpm. The solution was neutralized using 0.12M NaH2PO4 / 0.4% Tween 20. The supernatant was transferred to an ELISA plate and Aβ40 and 42 were measured. Readings were quantified by comparison with Aβ40 and 42 standard curve ELISA. The ELISA was performed according to the MSD 96-well MULTI_SPOT human / rodent 4G8 Aβ Triplex Ultra-Sensitive assay (Meso Scale Discovery). The results in Table 10 show the binding of Aβ40 and 42 to PSR1 beads with increasing amounts of beads in pg / ml and percent overall. Very little binding of Aβ40 is evident at all bead concentrations. The amount detected corresponds to less than 1% of the total Aβ40 present in normal CSF. The binding of Aβ42 is apparent and represents 1-5% of all Aβ42 present in normal CSF.

  The observed binding may indicate the presence of oligomeric Aβ in normal CSF.

または、これらの知見は、ＰＳＲ１がモノマー性Ａβを低い親和性で結合できることを示唆し得る。

Alternatively, these findings may suggest that PSR1 can bind monomeric Aβ with low affinity.

Example 15 Binding of Aβ42 monomer to PSR1 can be blocked by low concentrations of plasma (FIG. 17))
The affinity of PSR1 binding to monomeric Aβ and aggregated Aβ was assessed by examining the effect of increasing plasma concentrations.

  To test the block of monomeric Aβ binding to PSR1, the beads were incubated with a large excess of monomeric Aβ42 (25 ng / ml) in the presence of increasing plasma concentrations (FIG. 17, triangles). As the plasma concentration increased, the amount of monomer binding decreased. 20 percent plasma inhibited more than 90% of PSR1 binding to monomer.

  To test the block of aggregated Aβ binding to PSR1, the beads were incubated with 200 mL / mL 5% AD brain homogenate in the presence of increasing plasma concentrations (FIG. 17, circles). In contrast to monomeric Aβ, up to 85% of plasma did not affect the binding of aggregated Aβ.

  These findings

は、モノマー性Ａβは、低い親和性でＰＳＲ１を結合するが（これは、他のタンパク質によってブロックされる可能性がある）、凝集ＡβはＰＳＲ１をより高い親和性で結合することを示唆している。

Suggests that monomeric Aβ binds PSR1 with low affinity (which may be blocked by other proteins), but aggregated Aβ binds PSR1 with higher affinity. Yes.

(Example 16 HCl dissociates the binding between aggregated tau protein and PSR1-beads (FIG. 18))
In order to perform efficient immunodetection of PSR1 binding aggregates, the aggregates should be eluted and dissociated into protein monomers compatible with ELISA. The chaotropic intensity of dissociation depends on the physicochemical properties of the aggregate. To optimize the dissociation of aggregated tau from PSR1, the following acidic conditions

を試験した。

Was tested.

Brain homogenates (BH) from normal or Alzheimer's disease (AD) were spiked into TBSTT (Tris buffered saline containing 1% Tween 20 and 1% Triton-X 100). 100 uL of each sample was mixed with 3 uL of PSR1-beads (30 mg / mL). The resulting mixture was incubated at 37 ° C. for 1 hour with constant shaking at 750 rpm. Unbound sample material was removed by washing the beads 6 times with TBST (Tris buffered saline containing 0.05% Tween 20). For each wash, after addition of TBST, beads were collected using magnetic force to remove TBST. The binding between the aggregated tau and the beads was dissociated under various conditions (shown in the table) while constantly shaking at 750 rpm. The dissociation reaction was placed on three separate plates at three different temperatures. 6M GdnSCN was diluted with H ₂ O and HCl was neutralized to pH 7.5 with NaOH. The supernatant was transferred to the same magnetically ELISA plate. Tau was quantified by human tau (total) ELISA (Biosource).

  The results are shown in FIG. The results show that 0.25N HCl at room temperature (RT) and 50 ° C. dissociates the aggregated tau from AD BH and PSR1-beads. These dissociation conditions have the same efficiency as 6M GdnSCN at RT (6M GdnSCN at RT is standard because it dissociates aggregated proteins without damaging the protein). Dissociation with 0.25 N HCl at 80 ° C. removed the tau signal of the ELISA, which is probably due to damage of the tau protein. The same dissociation efficiency as 6M GdnSCN could not be achieved with 0.1N HCl-glycine containing 150 mM NaCl at pH 2.3. In normal BH, the signal was at the same level in all dissociation conditions. Condition 3 (30 minutes with 0.25N HCl at room temperature while shaking at 750 rpm) is a preferred dissociation condition for aggregated tau from PSR1 beads.

Example 17 Detection limit of PSR1 bead pull-down assay for total tau, P-tau 231 and P-tau 181 in normal human CSF spiked with AD BH (FIGS. 19A-F)
(All tau detection limits)
Alzheimer's disease brain homogenate (AD BH) was spiked into normal human CSF. 200 uL of each sample was mixed with 50 uL of 5 × TBSTT (Tris buffered saline containing 1% Tween 20 and 1% Triton-X 100) and 12 uL of PSR1-beads (30 mg / mL). The resulting mixture was incubated at 37 ° C. for 1 hour with constant shaking at 550 rpm. Unbound sample material was removed by washing the beads 6 times with TBST (Tris buffered saline containing 0.05% Tween 20). For each wash, after addition of TBST, beads were collected using magnetic force to remove TBST. The bead-bound aggregated tau was dissociated with 0.25N HCl for 30 minutes at room temperature with constant shaking at 750 rpm. The solution was neutralized using 0.25N NaOH. The supernatant was transferred to a magnetic INNOTEST hTAU Ag ELISA plate. The INNOTEST hTAU Ag ELISA was changed to 175 uL per reaction to synthesize the final sample volume from the PSR1-bead pull-down assay.

  The assay detection limit was determined using an S / N (signal to assay background) ratio of 2 or greater. The ELISA assay background was considered as signal with buffer only. The background of the pull-down assay was taken as the signal of CSF that did not spike AD BH. The detection limit for the 175 uL INNOTEST hTAU Ag ELISA was 0.032 fmol or 0.32 pM per assay. The detection limit of the tau PSR1 bead pull-down assay was 0.038 fmol, ie 0.19 pM per assay.

(P-tau 231 detection limit)
Alzheimer's disease brain homogenate (AD BH) was spiked into normal human CSF. 70 uL of each sample was mixed with 30 uL of 3.3 × TBSTT (Tris buffered saline containing 1% Tween 20 and 1% Triton-X 100) and 3 uL of PSR1-beads (30 mg / mL). The resulting mixture was incubated at 37 ° C. for 1 hour with constant shaking at 750 rpm. Unbound sample material was removed by washing the beads 6 times with TBST (Tris buffered saline containing 0.05% Tween 20). For each wash, after addition of TBST, beads were collected using magnetic force to remove TBST. The bead-bound aggregated tau was dissociated with 0.25N HCl for 30 minutes at room temperature with constant shaking at 750 rpm. The solution was neutralized using 0.25N NaOH. The supernatant was transferred to magnetic human tau [pT231] ELISA plates (Biosource).

  The assay detection limit was determined using an S / N (signal to assay background) ratio of 2 or greater. The ELISA assay background was considered as signal with buffer only. The background of the pull-down assay was taken as the signal of CSF that did not spike AD BH. The detection limit of human tau [pT231] ELISA was 0.09 fmol, ie 1.72 pM per assay. The detection limit of the tau [pT231] pull-down assay was 0.20 fmol, or 2.71 pM per assay.

(P-tau 181 detection limit)
Alzheimer's disease brain homogenate (AD BH) was spiked into normal human CSF. 70 uL of each sample was mixed with 30 uL of 3.3 × TBSTT (Tris buffered saline containing 1% Tween 20 and 1% Triton-X 100) and 3 uL of PSR1-beads (30 mg / mL). The resulting mixture was incubated at 37 ° C. for 1 hour with constant shaking at 750 rpm. Unbound sample material was removed by washing the beads 6 times with TBST (Tris buffered saline containing 0.05% Tween 20). For each wash, after addition of TBST, beads were collected using magnetic force to remove TBST. The bead-bound aggregated tau was dissociated with 0.25N HCl for 30 minutes at room temperature with constant shaking at 750 rpm. The solution was neutralized using 0.25N NaOH. The supernatant was transferred to a magnetic INNOTEST PHOSPHHO-TAU ₍ 181P ₎ ELISA plate.

The assay detection limit was determined using an S / N (signal to assay background) ratio of 2 or greater. The ELISA assay background was considered as signal with buffer only. The background of the pull-down assay was taken as the signal of CSF that did not spike AD BH. The detection limit of the INNOTEST PHOSPHO-TAU ₍ 181P ₎ ELISA was 0.04 fmol or 0.54 pM per assay. The detection limit of the tau [pT231] pull-down assay was 0.03 fmol, ie 0.44 pM per assay.

Claims (46)

A method for detecting the presence of a non-prion pathogenic conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer and, if present, binding of the reagent to the non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex;
If the non-prion pathogenic conformer is present in the sample, detecting the non-prion pathogenic conformer by its binding to the pathogenic conformer-specific binding reagent;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and interacts preferentially with the pathogenic prion protein. 2. The method of claim 1, wherein the non-prion pathogenic conformer is a conformer associated with amyloid disease. 3. The method of claim 2, wherein the amyloid disease is selected from the group consisting of systemic amyloidosis, tauopathy, and synucleinopathy. The non-prion pathogenic conformer is a conformer associated with a disease selected from the group consisting of Alzheimer's disease, ALS, immunoglobulin-related disease, serum amyloid A-related disease, and type II diabetes. Item 2. The method according to Item 1. 2. The method of claim 1, wherein the non-prion pathogenic conformer is an Alzheimer's disease conformer. 6. The method of claim 5, wherein the Alzheimer's disease conformer is amyloid beta (Aβ) protein. 6. The method of claim 5, wherein the Alzheimer's disease conformer is a tau protein. The pathogenic conformer-specific binding reagent is PrP _19-30 (SEQ ID NO: 242), PrP _23-30 (SEQ ID NO: 243), PrP _100-111 (SEQ ID NO: 244), PrP _101-110 (SEQ ID NO: _{245), PrP 154-165} (SEQ ID NO: _{246), PrP 226-237} (SEQ ID NO: 247), SEQ ID NO: 14, SEQ ID NO: 50, SEQ ID NO: 68,

7. The method of claim 6, wherein the method is derived from a compound selected from the group consisting of: The sample is an organ, whole blood, blood fraction, blood component, plasma, platelets, serum, cerebrospinal fluid (CSF), brain tissue, nervous system tissue, muscle tissue, bone marrow, urine, tear, non-neural system tissue, 2. The method of claim 1, wherein the method is selected from the group consisting of biopsy or autopsy. The method of claim 1, wherein the sample comprises plasma or cerebrospinal fluid. The prion protein fragments are PrP _19-30 (SEQ ID NO: 242), PrP _23-30 (SEQ ID NO: 243), PrP _100-111 (SEQ ID NO: 244), PrP _101-110 (SEQ ID NO: 245), PrP _154-165 ( The method of claim 1, wherein the method is selected from the group of peptides consisting of SEQ ID NO: 246), PrP _226-237 (SEQ ID NO: 247), SEQ ID NO: 14, SEQ ID NO: 50 and SEQ ID NO: 68. The prion protein fragments are PrP _19-30 (SEQ ID NO: 242), PrP _23-30 (SEQ ID NO: 243), PrP _100-111 (SEQ ID NO: 244), PrP _101-110 (SEQ ID NO: 245), SEQ ID NO: 14, 2. The method of claim 1, wherein the method is selected from the group consisting of SEQ ID NO: 50 and SEQ ID NO: 68. The pathogenic conformer specific binding reagent consists of SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 14, SEQ ID NO: 50 and SEQ ID NO: 68. 2. The method of claim 1 comprising an amino acid sequence selected from the group. The pathogenic conformer-specific binding reagent has an amino acid sequence selected from the group consisting of SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 14, SEQ ID NO: 50, and SEQ ID NO: 68. The method of claim 1 comprising. The pathogenic conformer-specific binding reagent comprises:
(A) SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, or 241;
(B) SEQ ID NO: 229, 230, 232, 233, 237, 238, 239, or 240;
(C) SEQ ID NO: 230, 237, 238, 239, or 240;
(D) SEQ ID NO: 240;
(E)

The method of claim 1, comprising a peptoid reagent selected from the group consisting of: 2. The method of claim 1, wherein the pathogenic conformer specific binding reagent has a net positive charge of at least 3 at physiological pH. 17. The method of claim 16, wherein the reagent has a net positive charge of at least 4 at physiological pH. 2. The method of claim 1, wherein the pathogenic conformer specific binding reagent is detectably labeled. The method of claim 18, wherein the reagent is detectably labeled with biotin. The method of claim 1, wherein the reagent is bound to a solid support. 21. The method of claim 20, wherein the solid support is selected from the group consisting of nitrocellulose, polystyrene latex, polyvinyl fluoride, diazotized paper, nylon membrane, activated beads, and magnetically responsive beads. A method for detecting the presence of a non-prion pathogenic conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer and, if present, binding of the reagent to the non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex;
Contacting the complex with a conformation disease protein-specific binding reagent under conditions that permit binding;
If the non-prion pathogenic conformer is present in the sample, detecting the presence of the non-prion pathogenic conformer by its binding to the conformation disease protein-specific binding reagent;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and interacts preferentially with the pathogenic prion protein. 23. The method of claim 22, wherein the method further comprises removing unbound sample material after formation of the complex. 23. The method of claim 22, wherein the conformational disease protein specific binding reagent is a labeled antibody. 23. The method of claim 22, wherein the non-prion pathogenic conformer is an Aβ protein and the conformation disease protein specific binding reagent is an anti-Aβ antibody. A method for detecting the presence of a non-prion pathogenic conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer and, if present, binding of the reagent to the non-prion pathogenic conformer. Contacting under the enabling conditions to form a first complex;
Removing unbound sample material;
Dissociating the non-prion pathogenic conformer from the first complex, thereby providing a dissociated non-prion pathogenic conformer;
Contacting the dissociated non-prion pathogenic conformer with a first conformational disease protein-specific binding reagent under conditions that permit binding to form a second complex;
If the non-prion pathogenic conformer is present in the sample, detecting the presence of the non-prion pathogenic conformer by detecting the formation of the second complex;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and interacts preferentially with the pathogenic prion protein. 27. The method of claim 26, wherein formation of the second complex is detected by using a detectably labeled second conformational disease protein specific binding reagent. 27. The method of claim 26, wherein the pathogenic conformer specific reagent is coupled to a solid support. 27. The method of claim 26, wherein the first conformational disease protein-specific binding reagent is coupled to a solid support. 27. The method of claim 26, wherein the non-prion pathogenic conformer is dissociated from the first complex by exposing the first complex to guanidine thiocyanate. 27. The method of claim 26, wherein the non-prion pathogenic conformer is dissociated from the first complex by exposing the complex to high or low pH. 32. The method of claim 31, further comprising neutralizing a high pH or a low pH after the dissociation. 27. The method of claim 26, wherein the non-prion pathogenic conformer is Aβ protein and the conformation disease protein specific binding reagent is an anti-Aβ antibody. 34. The method of claim 33, wherein the Aβ protein is dissociated from the first complex by exposing the complex to high pH conditions. 35. The method of claim 34, wherein the high pH condition is about 0.1 N NaOH at about 80 ° C. A method for detecting the presence of a non-prion pathogenic conformer comprising:
A first pathogenic conformer-specific binding reagent is present in a sample suspected of containing the non-prion pathogenic conformer, and the first to the non-prion pathogenic conformer, if present. Contacting under conditions that allow binding of one reagent to form a first complex;
To the sample suspected of containing the non-prion pathogenic conformer, a second pathogenic conformer-specific binding reagent comprising a detectable label is added to the sample in the first complex. Contacting under conditions that allow binding of the second reagent to a non-prion pathogenic conformer;
If the non-prion pathogenic conformer is present in a sample, detecting the non-prion pathogenic conformer by its binding to the second reagent;
Wherein the first and second pathogenic conformer-specific binding reagents are derived from prion protein fragments and interact preferentially with pathogenic prion protein. A method for detecting the presence of a non-prion pathogenic conformer comprising:
(A) Conformation disease protein-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer, and if present, binding of the reagent to the non-prion pathogenic conformer Contacting to form a complex under conditions that allow:
(B) removing unbound sample material;
(C) binding of the pathogenic conformer specific binding reagent comprising a detectable label to the complex and the pathogenic conformer specific binding reagent to the non-prion pathogenic conformer Contacting under conditions that allow for;
If the non-prion pathogenic conformer is present in the sample, detecting the non-prion pathogenic conformer by its binding to the pathogenic conformer-specific binding reagent;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and interacts preferentially with the pathogenic prion protein. A method for detecting the presence of a non-prion pathogenic conformer comprising:
Providing a solid support comprising a pathogenic conformer specific binding reagent;
Binding a detectably labeled ligand to the solid support, the binding affinity of the pathogenic conformer-specific binding reagent to the detectably labeled ligand being determined by the non-reactivity of the reagent. Weaker than the binding affinity for the prion pathogenic conformer, and step;
Binding the solid support to a sample under conditions that allow the non-prion pathogenic conformer to bind to the reagent and displace the ligand when present in the sample;
Detecting a complex formed between the reagent and the non-prion pathogenic conformer from the sample;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and preferentially interacts with the pathogenic prion protein. A method for distinguishing between a non-prion pathogenic conformer and a non-prion non-pathogenic conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer and, if present, binding of the reagent to the non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex;
Distinguishing the non-prion pathogenic conformer from the non-prion non-pathogenic conformer by binding of the pathogenic conformer to the reagent;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and interacts preferentially with the pathogenic prion protein. A method for diagnosing non-prion conformation disease,
Allows binding of pathogenic conformer-specific binding reagents to samples suspected of containing non-prion pathogenic conformers, and if present, to the non-prion pathogenic conformers Contacting under conditions to form a complex;
If the non-prion pathogenic conformer is present in the sample, detecting the non-prion pathogenic conformer by its binding to the reagent;
Diagnosing conformational disease when said non-prion pathogenic conformer is detected;
Wherein the pathogenic conformer-specific binding reagent is derived from a prion protein fragment and interacts preferentially with the pathogenic prion protein. A method for detecting the presence of a non-prion pathogenic conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer and, if present, binding of the reagent to the non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex;
If the non-prion pathogenic conformer is present in the sample, detecting the non-prion pathogenic conformer by its binding to the pathogenic conformer-specific binding reagent;
Wherein the pathogenic conformer-specific binding reagent comprises a peptoid region comprising SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, or 241. Method. A method for detecting the presence of a non-prion pathogenic conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the non-prion pathogenic conformer and, if present, binding of the reagent to the non-prion pathogenic conformer. Contacting under the enabling conditions to form a complex;
If the non-prion pathogenic conformer is present in the sample, detecting the non-prion pathogenic conformer by its binding to the pathogenic conformer-specific binding reagent;
Wherein the pathogenic conformer specific binding reagent comprises:

A method selected from. A method for detecting the presence of a pathogenic Alzheimer's disease conformer comprising:
A pathogenic conformer-specific binding reagent in a sample suspected of containing the pathogenic Alzheimer's disease conformer and, if present, binding of the reagent to the pathogenic Alzheimer's disease conformer. Contacting under the enabling conditions to form a complex;
Contacting the complex with a conformation disease protein-specific binding reagent under conditions that permit binding;
If the pathogenic Alzheimer's disease conformer is present in the sample, detecting the presence of the pathogenic Alzheimer's disease conformer by its binding to the conformation disease protein-specific binding reagent;
And the pathogenic conformer-specific binding reagent comprises

Is that way. 44. The method of claim 43, wherein the pathogenic Alzheimer's disease conformer is an Aβ protein and the conformation disease protein-specific binding reagent is an anti-Aβ antibody. 44. The method of claim 43, wherein the pathogenic Alzheimer's disease conformer is a tau protein and the conformation disease protein-specific binding reagent is an anti-tau antibody. 44. The method of claim 43, wherein the pathogenic conformer specific binding reagent is coupled to magnetic beads.

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